Pharmaceutical compositions

ABSTRACT

The present invention relates to an ionic complex comprising a cationic polypeptide and an anionic excipient selected from: a PEG-carboxylic acid; a fatty acid having 10 or more carbon atoms; an anionic phospholipid; and a combination thereof. The invention also relates to a pharmaceutical composition comprising the ionic complex of the invention and a pharmaceutically acceptable carrier. The cationic polypeptide of the ionic complex has pharmacological activity and the complex can provide a more desirable pharmacokinetic profile for the cationic polypeptide of the complex as compared to the cationic polypeptide alone following administration. As such, the invention also relates to the use of the ionic complex and pharmaceutical composition comprising same to treat a subject suffering from a disease or disorder that is responsive to the cationic polypeptide of the ionic complex.

RELATED APPLICATION

This application is a continuation of U.S. application Ser. No.14/775,911, filed Sep. 14, 2015, which is a U.S. National PhaseApplication under 35 U.S.C. § 371 of International Application No.PCT/US2014/029421, filed Mar. 14, 2014, which claims the benefit of U.S.Provisional Application No. 61/792,440, filed Mar. 15, 2013, thecontents of which are incorporated herein by reference in its entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Oct. 7, 2014, isnamed R2054-7001WO_SL.txt and is 48,749 bytes in size.

BACKGROUND OF THE INVENTION

It is often desirable to enhance the pharmacokinetic characteristics ofan active agent (e.g., to extend the duration of drug action or tominimize any undesirable effects). Drugs, in particular peptidic drug,are typically readily soluble in the body and can be absorbed rapidlyresulting in a sudden burst of available drug as opposed to a moregradual release. Compositions that can provide for a more gradual orextended release of drug can result in reduced fluctuations inconcentration of the drug following administration, increased drugloading per administration, increased stability and efficacy both invivo and in vitro, reduced toxicity and increased patient compliance dueto less frequent administrations. As such, a need exists forpharmaceutical compositions containing an active agent that provide foran extended release of the active agent.

SUMMARY OF THE INVENTION

The present invention relates to an ionic complex comprising a cationicpolypeptide and an anionic excipient selected from: a PEG-carboxylicacid; a fatty acid having 10 or more carbon atoms; an anionicphospholipid; and a combination thereof in different molar ratios. Theinvention also relates to a pharmaceutical composition comprising theionic complex of the invention and a pharmaceutically acceptablecarrier. The cationic polypeptide of the ionic complex haspharmacological activity and the complex can provide a more desirablepharmacokinetic profile for instance as a sustained release formulation,for the cationic polypeptide of the complex as compared to the cationicpolypeptide alone, following administration.

The invention also relates to the use of the ionic complex andpharmaceutical composition comprising same to treat a subject sufferingfrom a disease or disorder that is responsive to the pharmacologicalactivity possessed by the cationic polypeptide of the ionic complex. Inone embodiment, the disorder to be treated is responsive to themodulation of the melanocortin-4 receptor (MC4R) in a subject in need oftreatment. The method comprises administering to the subject aneffective amount of an ionic complex comprising as the cationicpolypeptide an MC4R modulator such as those described in Formula Iherein. In a particular embodiment, the disorder responsive tomodulation of the MC4R includes type 1 diabetes, type 2 diabetes,obesity, insulin resistance, metabolic syndrome, male erectiledysfunction, female sexual disorder, non-alcoholic fatty liver disease,non-alcoholic steatohepatitis, disorders of substance abuse, includingalcoholism feeding disorders, cachexia, inflammation and anxiety.

In certain embodiments, the compounds and compositions of the presentinvention possess higher selectivity and potency for the MC4R andmelanocortin-3 receptor (MC3R) when compared to melanocortin-1 receptor(MC1R). The compounds and compositions of the present invention canreduce or eliminate such undesirable side effects as increase in bloodpressure effects, increase in heart rate, undesired effects on sexualarousal, and increase in skin pigmentation.

The ionic complex and pharmaceutical compositions of the presentinvention comprising same can enhance the pharmacokinecticcharacteristics of the cationic polypeptide of the complex. For example,the duration of pharmacological action of the cationic polypeptide canbe extended while significantly narrowing the maximal to minimal drugexposure ratios in its pharmacokinetic profile. The therapeutic dose ofthe cationic peptide therefore can be maintained within a beneficialexposure range in the body, thereby alleviating the possibility ofundesirable side effects that could result due to a high exposure of thecationic polypeptide drug alone. Compositions that can provide for amore gradual or extended release of active can result in reducedfluctuations in concentration of the active following administration,increased active loading per administration, increased stability andefficacy both in vivo and in vitro, reduced toxicity and increasedpatient compliance due to less frequent administrations. The ioniccomplex compositions of this invention are suitable for effectivetherapeutic administration over a diverse set of dosing ranges includingat least once daily, once weekly, once every 2 weeks, once every fourweeks, once every 2 months, once every 3 months, once every 4 months,once every 5 months or once every 6 months.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph showing the pharmacokinetic profile of each of theidentified pharmaceutical compositions following administration tocynomolgous monkeys.

FIG. 2 is a graph showing the pharmacokinetic profile of each of theidentified pharmaceutical compositions following administration tocynomolgous monkeys.

FIG. 3 is a graph showing the pharmacokinetic profile of each of theidentified pharmaceutical compositions following administration tocynomologous monkeys.

DETAILED DESCRIPTION OF THE INVENTION

A description of example embodiments of the invention follows.

Glossary

The nomenclature used to define the peptides is that typically used inthe art wherein the amino group at the N-terminus appears to the leftand the carboxyl group at the C-terminus appears to the right.

As used herein, the term “amino acid” includes both a naturallyoccurring amino acid and a non-natural amino acid. Unless otherwiseindicated, all amino acids and their residues found in the compoundsdescribed herein can be either in D or L configuration.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. All publications, patentapplications, patents and other references mentioned herein areincorporated by reference in their entirety.

Symbol Meaning Abu α-aminobutyric acid Ac acyl group Aibα-aminoisobutyric acid Ala or A alanine Arg or R arginine Asn or Nasparagine Asp or D aspartic acid Atc

2-aminotetralin-2-carboxylic acid Cha β-cyclohexylalanine sChp

1-amino-4-phenylcyclohexane-1-carboxylic acid Cys or C cysteine hCyshomocysteine Dbu 2,4-diaminobutyric acid Dpr 2,3-diaminopropionic acidGln or Q glutamine Glu or E glutamic acid Gly or G glycine His or Hhistidine Hyp hydroxyproline Ile or I isoleucine Leu or L leucine Lys orK lysine Met or M methionine 1-Nal (1-naphthyl)-alanine 2-Nal(2-naphthyl)-alanine Nle norleucine Orn ornithine Pen penicillamine Pheor F phenylalanine Pro or P proline QAla

quinolinylalanine or 2-amino-3-(quinolin- 3-yl)propanoic acid Sarsarcosine (N-methylglycine) Ser or S Serine Tle tert-leucine (tert-butylglycine) TzAla

3-(1,2,4-triazol-1-yl)-L-Ala Thr or T threonine Trp or W tryptopham Tyror Y tyrosine Val or V valine BHA benzhydrylamine Boctert-butyloxycarbonyl But tertiary butyl DIPEA N,N-diisopropylethylamineDTT dithiothreitol Fmoc fluorenylmethyloxycarbonyl HBTU2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphateMCR4 melanocortin-4 receptor Mtt 4-methyltrityl NMP N-methylpyrrolidoneOBut tertiary butoxy OPip 2-phenylisopropyl Pbf2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl Trt trityl TIStriisopropylsilane TFA trifluoroacetic acid

Unless otherwise indicated, all abbreviations (e.g. Ala) of amino acidsin this disclosure refer to amino acid residues, i.e. stand for thestructure of —NH—C(R)(R′)—CO—, wherein R and R′ each is, independently,hydrogen or the side chain of an amino acid (e.g., R═CH₃ and R′═H forAla, or R and R′ may be joined to form a ring system).

The designation “Ac” or “NH₂” at a terminus of a polypeptide indicatesthat the corresponding terminus is acylated or amidated, respectively.

The phrase “a covalent bond between amino acid side chains” means thatthe side chains of two amino acid residues in question each includes afunctional group capable of forming a covalent bond with one another.Examples of such bonds include disulfide bridges formed by Cys, hCys, orPen side chains, and amid bonds formed by an amino group of one aminoacid side chain and a carboxy group of another amino acid side chain,such as, e.g. Asp, Glu, Lys, Orn, Dbu, or Dpr. When a covalent bondbetween amino acid side chains is formed, the polypeptide may becomecyclized. Such a cyclic polypeptide may be indicated either by astructural formula or by using the short-hand notation “c( )” or“cyclco( )” For example, “-c(Cys-Cys)-” or “-cyclo(Cys-Cys)-” denotesthe structure:

while “-c(Asp-Lys)-” or “-cyclo(Asp-Lys)-” denotes the structure:

Ionic Complex:

The invention relates to an ionic complex comprising a cationicpolypeptide and an anionic excipient selected from: a PEG-carboxylicacid; a fatty acid having 10 or more carbon atoms; a phospholipid; and acombination thereof. The molar ratio of the cationic polypeptide to theanionic excipient in the ionic complex ranges from, for instance, about1:1 to about 1:10 wherein the molar ratio is based on a charge of thecationic polypeptide to charge of the anionic excipient. The molar ratiois frequently selected from about 1:1, about 1:2, about 1:3, about 1:4,about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, or about 1:10.

Anionic Excipients Anionic Phospholipid:

As used herein, an “anionic phospholipid is a phospholipid wherein oneor more oxygen atoms of the phosphate group(s) has been deprotonatedresulting in an organophosphate oxoanion and a negatively charged lipid.The naturally occurring anionic phospholipids typically comprise a C₁₆or larger fatty acid chain. The anionic phospholipid can bear one ormore negative charges (e.g., 1, 2, 3, 4, 5, 6 or more). In someembodiments the anionic phospholipid is selected from: a phosphatidicacid (PA), a phosphatidylglycerol (PG), a phosphatidylinositol (PI), ora phosphatidylserine (PS). Suitable anionic lipids include:L-α-phosphatidic acid, 1-oleoyl lysophophatidic acid,L-α-phosphatidylglycerol,1,2-di-O-tetradecyl-sn-glycero-3-phospho-(1′-rac-glycerol),1,2-dimyristoyl-sn-glycero-3-phospho-(1′-rac-glycerol) (DMPG),1,2-dimyristoyl-sn-glycero-3-phospho-L-serine (DMPS),1,2-dipalmitoyl-sn-glycero-3-phosphatidic acid (DPPA),1,2-distearoyl-sn-glycero-3-phosphatidic acid (DSPA),1,2-disteroyl-sn-glycero-3-phosphoethanolamine (DSPE), mPEG-2,000-DSPE,mPEG-5,000-DSPE, 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine(DPPE), mPEG-2,000-DPPE, mPEG-5,000-DPPE,1-(1,2-dihexadecanoylphosphatidyl)inositol-4,5-bisphosphate, trisodiumsalt, and 1-(1,2-dihexadecanoylphosphatidyl)inositol-3,4,5-triphosphate, tetrasodium salt,1-palmitoyl-2-oleoylphosphatidylglycerol (POPG),1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphoglycerol (PAPG), DSPG(Distearoyl Phosphatidyglycerol), DPPG (DipalmitoylPhosphatidyglycerol), DEPG (Dielaidoyl Phosphatidyglycerol), DOPG(Dioleoyl Phosphatidyglycerol), DEPA (Dielaidoy Phosphatidic Acid), DOPA(Dioleoyl Phosphatidic Acid), DSPS (Distearoyl Phosphatidylserine), DPPS(Dipalmitoyl Phosphatidylserine), DEPS (Dielaidoy Phosphatidylserine),and DOPS (Dioleoyl Phosphatidylserine), L-α-lysophosphatidylserine,L-α-lysophosphatidylinositol, tetradecylphosphonic acid,L-α-phosphatidylinositol-4-phosphate,L-α-phosphatidylinositol-4,5-bisphosphate,1,2-diphytanoyl-sn-glycero-3-phosphate,1,2-di-O-tetradecyl-sn-glycero-3-phospho-(1

rac-glycerol) and mixtures thereof.

Specific anionic phospholipids for use in the present invention are1,2-Distearoyl-sn-Glycero-3-Phosphoethanolamine (DSPE) conjugatedPolyethylene Glycol, the structure of which is as follows:

with the value of “n” varying with molecular weight. Such anionicphospholipids are referred to herein as mPEG-(Mol. Wt.)DSPE. Suitableexamples include, mPEG-2,000-DSPE and mPEG-5,000-DSPE wherein DSPE is1,2-disteroyl-sn-glycero-3-phosphoethanolamine. Other example includemPEG-2,000-DPPE and mPEG-5,000-DPPE wherein DPPE is1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine.

Synthetically anionic phospholides with chain lengths shorter than C₁₆can also be obtained and utilized. Synthetic lipids can also be obtainedwith fatty acids that provide for additional anionic groups in the fattyacid chains. For example, a1-palmitoyl-2-glutaryl-sn-glycero-3-phosphocholine which incorporates ashorter fatty acid chain that terminates in a carboxylate group.

These naturally occurring and synthetic anionic lipids may be obtainedfrom several commercial sources such as, Avanti polar lipids (Alabaster,Ala.), or Lipoid LLC (Newark, N.J.), or Cordon Pharma (Boulder, Colo.),or NOF Corp America (White Plains, N.Y.).

Fatty Acid:

As used herein, a “fatty acid” is a carboxylic acid with a longaliphatic tail (chain), which is either saturated or unsaturated and hasa carbon atom chain of 10 or more atoms. The carbon atom chain can be alinear, branched, saturated, mono or poly-unsaturated chain.

Suitable saturated fatty acids include, but are not limited to: capricacid, lauric acid, myristic acid, palmitic acid, stearic acid, arachidicacid, docosanoic acid, tetracosanoic acid, and hexacosanoic acid.

Suitable unsaturated fatty acids include, but are not limited to:cis-2-decenoic acid, myristoleic acid, palmitoleic acid, sapienic acid,oleic acid, elaidic acid, vaccenic acid, linoleic acid, linoelaidicacid, α-linolenic acid, arachidonic acid, cis-parinaric acid,eicosapentaenoic acid, and phytanic acid.

PEG-Carboxylic Acids:

As used herein, a “PEG-carboxylic acid” means a polyethylene glycol(PEG) polymer that has been functionalized with a carboxylic acid. ThePEG can be functionalized to be monofunctional (monocarboxylate) orhomobifunctional (dicarboxylate). The functionalized PEG can be linearor branched. The PEG-carboxylic acid suitable for use in the inventioncan have an average molecular weight range of from about 1,000 to about100,000.

Homobifunctional PEGs suitable for use in the invention can be generallydepicted as COOH-PEG-COOH, or by the chemical formula:

-   -   COOH—(CH₂CH₂O)_(n)—CH₂CH₂—COOH, or        COOH—CH₂CH₂—CO—O(CH₂CH₂O)_(n)—CH₂CH₂—COOH, or        COOH—CH₂CH₂—CO—O(CH₂CH₂O)_(n)—CO—CH₂CH₂—COOH        with the value of “n” varying with molecular weight. For        example, the PEG-carboxylic acid denoted in the examples as        PEG-10,000-dicarboxylate is a homobifunctional PEG having a        molecular weight of 10,000. An average molecular weight range of        from about 1,000 to about 100,000 is suitable. Typically,        molecular weights can range from 1,000-40,000

One type of monofunctional PEG-carboxylic acids suitable for use in theinvention can be generally depicted as mPEG-COOH, or by the chemicalformula: CH₃O—(CH₂CH₂O)_(n)—CH₂CH₂—COOH orCH₃O—(CH₂CH₂O)_(n)—CO—CH₂CH₂—COOH again with the value of “n” varyingwith molecular weight. For example, the mPEG-10000 used in the exampleswould a monofunctional PEG having a methoxy group as indicate in theformula. An average molecular weight range of from about 1,000 to about100,000 is suitable. Typically, molecular weights can range from1,000-40,000

Another type of monofunctional PEG-carboxylic acid suitable for use inthe invention can be generally depicted as PEG-COOH or by the chemicalformula: HO—(CH₂CH₂O)_(n)—CH₂CH₂—COOH with the value of “n” varying withmolecular weight. For example, the PEG-carboxylic acid denoted in theexamples as PEG-10,000-monocarboxylate is a monofunctional PEG having amolecular weight of 10,000. An average molecular weight range of fromabout 1,000 to about 100,000 is suitable. Typically, molecular weightscan range from 1,000-40,000

Suitable PEG-carboxylic acids include, but are not limited to,PEG-10,000-monocarboxylate, PEG-20,000-monocarboxylate,mPEG-1,000-monocarboxylate, mPEG-2,000-monocarboxylate,mPEG-5,000-monocarboxylate, mPEG-10,000-monocarboxylate,mPEG-20,000-monocarboxylate, mPEG-30,000-monocarboxylate,mPEG-40,000-monocarboxylate, PEG-1,000-dicarboxylate,PEG-2,000-dicarboxylate, PEG-3,500-dicarboxylate,PEG-5,000-dicarboxylate, PEG-7,500-dicarboxylate,PEG-10,000-dicarboxylate, and Y-shape PEG-40,000-monocarboxylate. TheY-Shaped PEG-Carboxylic Acids referred to above can be depicted asfollows:

again with the value of “n” determining the molecular weight.

In particular embodiments, the PEG-carboxylic acid is selected from:PEG-5,000-monocarboxylate, PEG-10,000-monocarboxylate,PEG-20,000-monocarboxylate, mPEG-5,000-monocarboxylate,mPEG-10,000-monocarboxylate, mPEG-20,000-monocarboxylate,PEG-5,000-dicarboxylate, and PEG-10,000-dicarboxylate.

Suitable PEG-carboxylic acids can be commercially obtained from Nanocs,Inc. (New York, N.Y.), JenKem Technology (Allen, Tex.), or NOF CorpAmerica (White Plains, N.Y.).

In certain embodiments, two or more of the anionic excipients describedabove (e.g., PEG-carboxylic acid, anionic phospholipid and fatty acid)can be employed in the formulation. For example, a PEG-carboxylic acidand an anionic phospholipid, a PEG-carboxylic acid and a fatty acid, ora fatty acid and an anionic phospholipid can be used in thepharmaceutical formulation of the invention. In yet another embodiment,three or more anionic excipients selected from a fatty acid, an anionicphospholipid, and a PEG-carboxylic acid are present in thepharmaceutical formulation. In particular embodiments, the combinationof anionic excipients is selected from: stearic acid andmPEG-2,000-DSPE; DPPA and PEG-10,000-dicarboxylate; DPPA and PEG-3,350;DPPA; DPPA and mPEG-3,350 and mPEG-2,000-DSPE. In yet anotherembodiment, any of the anionic pospholipids described herein can befurther combined with carboxymethyl cellulose (CMC), such as acombination of mPEG-2000-DSPE and CMC.

Cationic Peptides:

As used herein, “cationic polypeptide” means any polypeptide thatcarries a positive charge at a pH of about 5.0. The cationic polypeptidecan include naturally occurring amino acid residues, non-naturallyoccurring amino acid residues or a mixture thereof. The positive chargeof the cationic polypeptides results from a cationic functional groupwhich is present either in the side chain of an amino acid of thepolypeptide sequence, as part of the alpha amino group of an amino acidof the polypeptide sequence or both. The positive charge of the cationicpolypeptide can also result from a cationic functional group ligated tothe polypeptide at either of the termini of the peptide sequence or inthe side chain of an amino acid in the sequence. There can be more thanone cationic functional group present in the cationic polypeptide. Forexample, the cationic polypeptide can have 1, 2, 3, 4, 5, 6 or morecationic functional groups, each of which can provide a positive charge.Such functional groups include, for example, amino groups (primary,secondary or tertiary), quaternary ammonium groups, guanidino groups,amidino groups, pyridine groups, imidazole groups, phosphonium groupsand sulfonium groups. In a particular embodiment, the cationic group isan amino group, a guanidino group or an imidazole group.

The cationic polypeptides of the invention may possess one or morechiral centers and so exist in a number of stereoisomeric forms. Allstereoisomers and mixtures thereof are included in the scope of thepresent invention. Racemic compounds may either be separated usingpreparative HPLC and a column with a chiral stationary phase or resolvedto yield individual enantiomers utilizing methods known to those skilledin the art. In addition, chiral intermediate compounds may be resolvedand used to prepare chiral compounds of the invention.

The cationic polypeptides described herein may exist in one or moretautomeric forms. All tautomers and mixtures thereof are included in thescope of the present invention.

Cationic polypeptides suitable for use in the present invention include,but art not limited to the polypeptides categories and specificpolypeptides listed below and the polypeptides represented by Formula I.The cationic polypeptides are pharmacologically active.

LHRH (GnRH) agonists: Leuprolide: Pyr-His-Trp-Ser-Tyr-D-Leu-Leu-Arg-Pro-NHEt (SEQ ID NO: 46) Buserelin:Pyr-His-Trp-Ser-Tyr-D-Ser(tBu)- Leu-Arg-Pro-NHEt (SEQ ID NO: 47):Histrelin: Pyr-His-Trp-Ser-Tyr-D-His(Bzl)- Leu-Arg-Pro-NHEt(SEQ ID NO: 48) Goserelin: Pyr-His-Trp-Ser-Tyr-D-Ser(tBu)-Leu-Arg-Pro-Azagly-NH₂ (SEQ ID NO: 49) DeslorelinPyr-His-Trp-Ser-Tyr-D-Trp- Leu-Arg-Pro-NHEt (SEQ ID NO: 50) NafarelinPyr-His-Trp-Ser-Tyr-D-Nal(2)- Leu-Arg-Pro-Gly-NH₂ (SEQ ID NO: 51)Triptorelin Pyr-His-Trp-Ser-Tyr-D-Trp- Leu-Arg-Pro-Gly-NPh(SEQ ID NO: 52) Avorelin Pyr-His-Tip-Ser-Tyr-D-Tip(2-Me)-Leu-Arg-Pro-NHEt (SEQ ID NO: 53) GnRH antagonists: Abarelix:Ac-D-Nal(2)-D-Phe (4Cl)-D-Pal(3)-Ser- (Me)Tyr-D-Asn-Leu-Lys(iPr)-Pro-D-Ala-NH₂ (SEQ ID NO: 54) Cetrorelix:Ac-D-Nal(2)-D-Phe(4Cl)-D-Pal(3)- Ser-Tyr-D-Cit-Leu-Arg- Pro-D-Ala-NH₂(SEQ ID NO: 55) Degarelix: Ac-D-Nal(2)-D-Phe(4Cl)-D-Pal(3)-Ser-Phe(4-(4S)- hexahydro-2,6-dioxo-4- pyrimidinyl(carbonyl)amino)-D-Phe(4-guanidino)- Leu-Lys(iPr)-Pro-D-Ala-NH₂ (SEQ ID NO: 56)Ganirelix: Ac-D-Nal(2)-D-Cpa(4)-D-Pal(3)- Ser-Tyr-D-Harg(Et)₂-Leu-Harg(Et)₂-Pro-D-Ala-NH₂ (SEQ ID NO: 57) Somatostatin analogs: OctreotideD-Phe-[Cys-Phe-D-Trp- Lys-Thr-Cys]-Thr-ol (SEQ ID NO: 58) LanreotideD-Nal(2)-[Cys-Tyr-D-Trp- Lys-Val-Cys]-Thr-NH₂ (SEQ ID NO: 59) VapreotideD-Phe-[Cys-Tyr-D-Trp-Lys- Val-Cys]-Trp-NH₂ (SEQ ID NO: 60) PasireotideCyclo[-(4R- (Signifor) 4-(2-aminoethylcarbamoyloxy)-L-prolyl-L-phenylglycyl-D- tryptophyl-L-lysyl-4-O-benzyl-L-tyrosyl-L-phenylalanyl-] (SEQ ID NO: 60)

Other Peptides: Glucagon Amylin Pramlintide Insulin

Glucagon-like peptide-1 (GLP-1)a GLP-1 agonist

Exenatide

Parathyroid hormone (PTH)Adrenocorticotropic hormone (ACTH)Botulinum toxinan amyloid peptide

Cholecystikinin Calcitonin

a conotoxin

Prialt

a gastric inhibitory peptidean insulin-like growth factor, including recombinantly manufacturedIGF-1, such as Increlexa growth hormone releasing factoran anti-microbial factor

Glatiramer (Copaxone)

Hematide (peginesatide)

Nasiritide

ANF peptideAngiotensin peptide

ACTH

Human Growth Hormone (hGH), including recombinantly manufactured hGH

Melanocortin

An opioid peptide

Dynorphin Oxytocin

Oxytocin analog, including antagonistsVasopressin and analogs andSomatostatin and its analogs

Cationic Polypeptides of Formula I:

Cationic Polypeptides for use in the invention are those of Formula (I)or a pharmaceutically acceptable salt thereof:

or a pharmaceutically acceptable salt thereof,

wherein:

R¹ is H, or a C1-C6 acyl;

R² is, —NR³R⁴, or −OR⁵ wherein R³, R⁴, and R⁵ are each independently isH or a C1-C6 alkyl;

A¹ is an amino acid residue selected from Arg, Lys, Orn, His, Nle, Phe,Val, Leu, Trp, Tyr, Ala, Ser, Thr, Gln, Asn, Asp, Glu, or TzAla; or

A¹ is a moiety selected from an optionally substituted C1-C12 alkyl, anoptionally substituted C6-C18 aryl, an optionally substituted C5-C18heteroaryl, an aralkyl wherein the aryl portion is an optionallysubstituted C6-C18 aryl, and the alkyl portion is an optionallysubstituted C1-C12 alkyl, or a heteroaralkyl, wherein the heteroarylportion is an optionally substituted C5-C18 heteroaryl, and the alkylportion is an optionally substituted C1-C12 alkyl;

A² and A⁸ is each independently an amino acid residue selected from Cys,hCys, Pen, Asp, Glu, Lys, Orn, Dbu, or Dpr, wherein A² and A⁸ arepairwise selected so as to be able to form covalent bond between theirrespective side chains;

A³ is absent or is an amino acid residue selected from Ala, Tle, Val,Leu, Ile, Cha, Pro, Ser, Thr, Lys, Arg, His, Phe, Gln, Sar, Gly, Asn, orAib;

A⁴ is absent or an amino acid residue selected from Atc, Ala, QAla, Aib,Sar, Ser, Thr, Pro, Hyp, Asn, Gln, an optionally substituted His, Trp,Tyr, Lys, Arg, sChp, or residue X, where the X is an amino acidrepresented by the following structural formula

A⁵ is an optionally substituted Phe, an optionally substituted 1-Nal, oran optionally substituted 2-Nal;

A⁶ is Arg; and

A⁷ is Trp,

wherein any amino acid residue is either in L- or in D-configuration.

In example embodiments, A³ and A⁴ are not both absent. Values andpreferred values of the remainder of the variables are as defined aboveand below with respect to Formula (I).

In example embodiments, when A⁴ is an amino acid, A³ is not Aib or Gly.Values and preferred values of the remainder of the variables are asdefined above with respect to Formula (I).

In example embodiments, when A⁴ is His and A⁵ is a D-Phe or 2-Nal, A³ isnot a D-amino acid or L-Ala. Values and preferred values of theremainder of the variables are as defined above with respect to Formula(I).

In example embodiments, when A² and A⁸ each is selected from Cys, hCysor Pen, then: (a) when A⁴ is absent, then A³ is not L-His; (b) when A³is absent, then A⁴ is not L-His; and (c) when A⁴ is His, then A³ is notGlu, Leu, or Lys. Values and preferred values of the remainder of thevariables are as defined above with respect to Formula (I).

In example embodiments: 1) A³ and A⁴ are not both absent; 2) when A⁴ isan amino acid, A³ is not Aib or Gly; and 3) when A⁴ is His and A⁵ is aD-Phe or 2-Nal, A³ is not a D-amino acid or L-Ala; 4) when A² and A⁸each is selected from Cys, hCys or Pen, then: (a) when A⁴ is absent,then A³ is not L-His; (b) when A³ is absent, then A⁴ is not L-His; and(c) when A⁴ is His, then A³ is not Glu, Leu, or Lys. Values andpreferred values of the remainder of the variables are as defined abovewith respect to Formula (I).

In another embodiment, the polypeptides of Formula (I), A⁴ is an L-aminoacid. In yet other embodiments, A⁴ is absent. Values and preferredvalues of the remainder of the variables are as defined above withrespect to Formula (I).

In example embodiments, A⁵ can be an optionally substituted 1-Nal or anoptionally substituted 2-Nal, for example, an optionally substitutedD-2-Nal. A⁵ can be substituted at any of the five aromatic carbons witha substituent selected from F, Cl, Br, I, —CH₃, —OH, —CN, amine, —NO₂,or —OCH₃.

In a further embodiment, the polypeptides of Formula (I), A⁵ is anoptionally substituted D-Phe. A⁵ can be substituted at any of the fivearomatic carbons with a substituent selected from F, Cl, Br, I, —CH₃,—OH, —CN, amine, —NO₂, or —OCH₃. Suitable examples of A⁵ include, butare not limited to, a D-amino acid residue selected from: Phe,Phe(2′-F), Phe(2′-Cl), Phe(2′-Br), Phe(2′-I), Phe(2′-CN), Phe(2′-CH₃),Phe(2′-OCH₃), Phe(2′-CF₃), Phe(2′-NO₂), Phe(3′-F), Phe(3′-C1),Phe(3′-Br), Phe(3′-I), Phe(3′-CN), Phe(3′-CH₃), Phe(3′-OCH₃),Phe(3′-CF₃), Phe(3′-NO₂), Phe(4′-F), Phe(4′-Cl), Phe(4′-Br), Phe(4′-I),Phe(4′-CN), Phe(4′-CH₃), Phe(4′-OCH₃), Phe(4′-CF₃), Phe(4′-NO₂),Phe(4′-t-Bu), Phe(2′,4′-diF), Phe(2′,4′-diCl), Phe(2′,4′-diBr),Phe(2′,4′-diI), Phe(2′,4′-di-CN), Phe(2′,4′-di-CH₃), Phe(2′,4′-di-OCH₃),Phe(3′,4′-diF), Phe(3′,4′-diCl), Phe(3′,4′-diBr), Phe(3′,4′-diI),Phe(3′,4′-di-CN), Phe(3′,4′-di-CH₃), Phe(3′,4′-di-OCH₃), Phe(3′,5′-diF),Phe(3′,5′-diCl), Phe(3′,5′-diBr), Phe(3′,5′-diI), Phe(3′, 5′-di-CN),Phe(3′,5′-diCH₃), Phe(3′,5′-di-OCH₃), or Phe(3′,4′,5′-triF). Values andpreferred values of the remainder of the variables are as defined abovewith respect to Formula (I).

In a further embodiment, the polypeptides of Formula (I), A⁵ is anoptionally substituted D-2-Nal. A⁵ can be substituted at any of the fivearomatic carbons with a substituent selected from F, Cl, Br, I, —CH₃,—OH, —CN, amine, —NO₂, or —OCH₃.

In yet another embodiment, the polypeptides of Formula (I), A⁴ is His,optionally substituted at any substitutable position with a substituentselected from F, Cl, Br, I, —CH₃, —OH, —CN, amine, —NO₂, benzyl or—OCH₃. Values and preferred values of the remainder of the variables areas defined above with respect to Formula (I).

In a particular embodiment, the compounds of the present invention arethose polypeptides of Formula (I) that possess EC₅₀ with respect to MC4Rfrom about 0.01 nM to about 10 nM, for example 0.01-3 nM, whilepossessing the ratio of EC₅₀(MC1R)/EC₅₀(MC4R) of at least 10.

In another embodiment, the polypeptides of the present invention includea polypeptide represented by any one of the following structuralformulas:

(SEQ ID NO: 1)

(SEQ ID NO: 43)

(SEQ ID NO: 44)

(SEQ ID NO: 45)

SEQ ID NO: 62

SEQ ID NO: 14

SEQ ID NO: 15

SEQ ID NO: 15

SEQ ID NO: 63

SEQ ID NO: 64

SEQ ID NO: 10

SEQ ID NO: 9

SEQ ID NO: 8

SEQ ID NO: 5

SEQ ID NO: 34

SEQ ID NO: 65

SEQ ID NO: 66

or a pharmaceutically acceptable salt thereof.

In yet another embodiment, the polypeptides of the present inventioninclude any one of the following structural formulas:

SEQ ID NO: 21

SEQ ID NO: 20

SEQ ID NO: 67

SEQ ID NO: 33

SEQ ID NO: 34

SEQ ID NO: 68

SEQ ID NO: 36

SEQ ID NO: 69

SEQ ID NO: 70

SEQ ID NO: 71

SEQ ID NO: 72

or a pharmaceutically acceptable salt thereof.

In a further embodiment, the polypeptides of the present inventioninclude the polypeptide represented by any one of the followingstructural formulas:

SEQ ID NO: 73

SEQ ID NO: 74

SEQ ID NO: 75

SEQ ID NO: 76

SEQ ID NO: 77

SEQ ID NO: 78

SEQ ID NO: 79

SEQ ID NO: 80

SEQ ID NO: 81

SEQ ID NO: 82

SEQ ID NO: 83

or a pharmaceutically acceptable salt thereof.

In yet another embodiment, the polypeptides of the present inventioninclude a polypeptide represented by formula (I), wherein A⁴ is an aminoacid residue selected from Atc, Ala, QAla, Aib, Sar, Ser, Thr, Pro, Hyp,Asn, Gln, a substituted His, Trp, Tyr, Lys, Arg, sChp, or residue X.Examples of such peptides include peptides represented by any one of thefollowing structural formulas:

(SEQ ID NO: 6) Ac-Arg-cyclo[Cys-D-Ala-His (3-Me)-D-Phe-Arg-Trp-Cys]-NH₂;(SEQ ID NO: 7) Ac-Arg-cyclo[Cys-D-Ala-His (1-Me)-D-Phe-Arg-Trp-Cys]-NH₂;(SEQ ID NO: 8) Ac-Arg-cyclo[Cys-D-Ala-Trp- D-Phe-Arg-Trp-Cys]-NH₂;(SEQ ID NO: 9) Ac-Arg-cyclo[Cys-D-Ala-Gln- D-Phe-Arg-Trp-Cys]-NH₂;(SEQ ID NO: 10) Ac-Arg-cyclo[Cys-D-Ala-Asn- D-Phe-Arg-Trp-Cys]-NH₂;(SEQ ID NO: 11) Ac-Arg-cyclo[Cys-D-Ala-Arg- D-Phe-Arg-Trp-Cys]-NH;(SEQ ID NO: 12) Ac-Arg-cyclo[Cys-D-Ala-Tyr- D-Phe-Arg-Trp-Cys]-NH₂;(SEQ ID NO: 13) Ac-Arg-cyclo[Cys-D-Ala-D- Pro-D-Phe-Arg-Trp-Cys]-NH₂;(SEQ ID NO: 14) Ac-Arg-cyclo[Cys-D-Ala-Pro- D-Phe-Arg-Trp-Cys]-NH₂;(SEQ ID NO: 15) Ac-Arg-cyclo[Cys-D-Ala-Pro- D-Phe(p-F)-Arg-Trp-Cys]-NH₂;(SEQ ID NO: 16) Ac-Arg-cyclo[Cys-D-Ala-Atc- D-Phe-Arg-Trp-Cys]-NH₂;(SEQ ID NO: 17) Ac-Arg-cyclo[Cys-D-Ala-QAla- D-Phe-Arg-Trp-Cys]-NH₂;(SEQ ID NO: 18) Ac-Arg-cyclo[Cys-D-Ala-sChp- D-Phe-Arg-Trp-Cys]-NH₂; or(SEQ ID NO: 19) Ac-Arg-cyclo[Cys-D-Ala-X-D- Phe-Arg-Trp-Cys]-NH₂,

or a pharmaceutically acceptable salt thereof.

In example embodiments, the polypeptides of the present inventioninclude a polypeptide represented by any one of the following structuralformulas:

(SEQ ID NO: 20) Ac-Arg-cyclo[hCys-Ala-D- Phe-Arg-Trp-Cys]-NH₂;(SEQ ID NO: 21) Ac-Arg-cyclo[hCys-D-Ala- D-Phe-Arg-Trp-Cys]-NH₂;(SEQ ID NO: 22) Ac-Arg-cyclo[hCys-D-Ala- D-Phe-Arg-Trp-Pen]-NH₂;(SEQ ID NO: 23) Ac-Arg-cyclo[Glu-D-Ala- D-Phe-Arg-Trp-Dpr]-NH₂;(SEQ ID NO: 24) Ac-Arg-cyclo[Glu-Ala-D- Phe-Arg-Trp-Dpr]-NH₂;(SEQ ID NO: 25) Ac-Arg-cyclo[hCys-Aib-D- Phe-Arg-Trp-Cys]-NH₂;(SEQ ID NO: 26) Ac-Arg-cyclo[hCys-Sar-D- Phe-Arg-Trp-Cys]-NH₂;(SEQ ID NO: 27) Ac-Arg-cyclo[hCys-Val-D- Phe-Arg-Trp-Cys]-NH₂;(SEQ ID NO: 28) Ac-Arg-cyclo[hCys-D-Val- D-Phe-Arg-Trp-Cys]-NH₂;(SEQ ID NO: 29) Ac-Arg-cyclo[hCys-Gln-D- Phe-Arg-Trp-Cys]-NH₂;(SEQ ID NO: 30) Ac-Arg-cyclo[hCys-D-Gln- D-Phe-Arg-Trp-Cys]-NH₂;(SEQ ID NO: 31) Ac-Arg-cyclo[hCys-Ala-D- Phe-Arg-Trp-Pen]-NH₂;(SEQ ID NO: 32) Ac-Arg-cyclo[D-Pen-D-Ala- D-Phe-Arg-Trp-hCys]-NH₂;(SEQ ID NO: 33) Ac-Arg-cyclo[Cys-D-Ala-D- Phe-Arg-Trp-hCys]-NH₂;(SEQ ID NO: 34) Ac-Arg-cyclo[Pen-D-Ala-D- Phe-Arg-Trp-hCys]-NH₂;(SEQ ID NO: 35) Ac-Arg-cyclo[D-hCys-D-Ala- D-Phe-Arg-Trp-Cys]-NH₂;(SEQ ID NO: 36) Ac-Arg-cyclo[hCys-Pro-D- Phe-Arg-Trp-Cys]-NH₂; or(SEQ ID NO: 37) Ac-Arg-cyclo[hCys-D-Pro- D-Phe-Arg-Trp-Cys]-NH₂,or a pharmaceutically acceptable salt thereof.

In another embodiment, the polypeptides of the present invention includepolypeptides represented by formula (I), wherein A³ is an amino acidresidue selected from Tle, Val, Leu, Ile, Cha, Pro, Ser, Thr, Lys, Arg,His, Phe, Gln, Sar, Gly, Asn, or Aib; and A⁴ is an amino acid residueselected from Atc, Ala, QAla, Aib, Sar, Ser, Thr, Pro, Hyp, Asn, Gln, asubstituted His, Trp, Tyr, Lys, Arg, sChp, or residue X. Examples ofsuch polypeptides are polypeptides represented by any one of thefollowing structural formulas:

(SEQ ID NO: 38) Ac-Arg-cyclo[Cys-Val-Gln-D- Phe-Arg-Trp-Cys]-NH₂;(SEQ ID NO: 39) Ac-Arg-cyclo[Cys-D-Val-Gln-D- Phe-Arg-Trp-Cys]-NH₂; or(SEQ ID NO: 40) Ac-Arg-cyclo[Cys-D-Va1-His (1-Me)-D-Phe-Arg-Trp-Cys]-NH₂,or a pharmaceutically acceptable salt thereof.

In a further embodiment, the polypeptides of the present inventioninclude a polypeptide represented by any one of the following structuralformulas:

(SEQ ID NO: 41) Ac-TzAla-cyclo[Cys-Ala-Gln-D- Phe-Arg-Trp-Cys]-NH₂; or(SEQ ID NO: 42) Ac-Glu-cyclo[Cys-Ala-His-D- Phe-Arg-Trp-Cys]-NH₂,or a pharmaceutically acceptable salt thereof.

In yet another embodiment, the polypeptides of the present inventioninclude a polypeptide represented by any one of the following structuralformulas:

(SEQ ID NO: 7) Ac-Arg-cyclo[Cys-D-Ala-His(1-Me)- D-Phe-Arg-Trp-Cys]-NH₂(SEQ ID NO: 9) Ac-Arg-cyclo[Cys-D-Ala-Gln-D-Phe- Arg-Trp-Cys]-NH₂(SEQ ID NO: 10) Ac-Arg-cyclo[Cys-D-Ala-Asn-D-Phe- Arg-Trp-Cys]-NH₂or a pharmaceutically acceptable salt thereof.

In a further embodiment, the polypeptides of the present inventioninclude a polypeptide represented by any one of the following structuralformulas:

(SEQ ID NO: 2) Ac-Arg-cyclo[Cys-D-Leu-His-D- Phe-Arg-Trp-Cys]-NH₂;(SEQ ID NO: 3) Ac-Arg-cyclo[Cys-D-Ile-His-D- Phe-Arg-Trp-Cys]-NH₂;(SEQ ID NO: 4) Ac-Arg-cyclo[Cys-D-Tle-His-D- Phe-Arg-Trp-Cys]-NH₂;(SEQ ID NO: 5) Ac-Arg-cyclo[Cys-D-Val-His-D- Phe-Arg-Trp-Cys]-NH₂,or a pharmaceutically acceptable salt thereof.

In a further embodiment, the polypeptides of the present inventioninclude a polypeptide represented by any one of the following structuralformulas:

(SEQ ID NO: 84) Ac-Arg-cyclo[Cys-D-Ala-His(1-Me)-D-2-Nal-Arg-Trp-Cys]-NH₂; (SEQ ID NO: 85)Ac-Arg-cyclo[Cys-D-Ala-Gln-D-2- Nal-Arg-Trp-Cys]-NH₂; or (SEQ ID NO: 86)Ac-Arg-cyclo[Cys-D-Ala-Asn-D-2- Nal-Arg-Trp-Cys]-NH₂,

or a pharmaceutically acceptable salt thereof. In a further eptides ofthe present invention include a polypeptide represented by any one ofthe following structural formulas:

(SEQ ID NO: 87) Ac-Arg-cyclo[Cys-D-Ala-His(1-Me)- D-Phe-Arg-Trp-Cys]-OH;(SEQ ID NO: 88) Ac-Arg-cyclo[Cys-D-Ala-Gln-D-Phe- Arg-Trp-Cys]-OH; or(SEQ ID NO: 89) Ac-Arg-cyclo[Cys-D-Ala-Asn-D-Phe- Arg-Trp-Cys]-OH,or a pharmaceutically acceptable salt thereof.In an alternative embodiment, the cationic peptides for use in theinvention are those of Formula (II) or a pharmaceutically acceptablesalt thereof:An isolated polypeptide of the following structural Formula (II):

or a pharmaceutically acceptable salt thereof,

wherein:

-   -   R¹ is —H, or a C1-C6 acyl;    -   R² is —NR³R⁴, or —OR⁵ wherein R³, R⁴, and R⁵ are each        independently is H or a C1-C6 alkyl;    -   A¹ is absent; or    -   A¹ is an amino acid residue selected from Arg, Lys, Orn, His,        Nle, Phe, Val, Leu, Trp, Tyr, Ala, Ser, Thr, Gln, Asn, Asp, Glu,        or TzAla; or    -   A¹ is a moiety selected from an optionally substituted C1-C12        alkyl, an optionally substituted C6-C18 aryl, an optionally        substituted C5-C18 heteroaryl, an aralkyl wherein the aryl        portion is an optionally substituted C6-C18 aryl, and the alkyl        portion is an optionally substituted C1-C12 alkyl, or a        heteroaralkyl, wherein the heteroaryl portion is an optionally        substituted C5-C18 heteroaryl, and the alkyl portion is an        optionally substituted C1-C12 alkyl;    -   A² and A⁸ is each independently an amino acid residue selected        from Cys, hCys, Pen, Asp, Glu, Lys, Orn, Dbu, or Dpr, wherein A²        and A⁸ are pairwise selected so as to be able to form covalent        bond between their respective side chains;    -   A³ is absent or is an amino acid residue selected from Ala, Tle,        Val, Leu, Ile, Cha, Pro, Ser, Thr, Lys, Arg, His, Phe, Gln, Sar,        Gly, Asn, Aib, or residue Y, wherein Y is an amino acid selected        from amino acids represented by the following structural        formulas

wherein:

-   -   R¹¹ and R¹², each independently, is H, —CH₃, phenyl, or benzyl;    -   R²¹, R²², R²³ and R²⁴, each independently is H, —CH₃, —CF₃,        phenyl, benzyl, F, Cl, Br, I, —OCH₃, or —OH;    -   R³¹, R³², R³³, R³⁴, R⁴¹, R⁴² and R⁴³, each independently is H,        —CH₃, —CF₃, phenyl, benzyl, F, Cl, Br, I, —OCH₃, or —OH;    -   A⁴ is absent or is an amino acid residue selected from Atc, Ala,        QAla, Aib, Sar, Ser, Thr, Pro, Hyp, Asn, Gln, an optionally        substituted His, Trp, Tyr, Lys, Arg, sChp, or residue X, where        the X is an amino acid selected from amino acids represented by        the following structural formulas:

wherein:

-   -   R⁵¹ and R⁵², each independently, is H, —CH₃, phenyl, or benzyl;    -   R⁶¹, R⁶², R⁶³, and R⁶⁴, each independently is H, —CH₃, —CF₃,        phenyl, benzyl, F, Cl, Br, I, —OCH₃, or —OH;    -   R⁷¹, R⁷², R⁷³, R⁷⁴, R⁸¹, R⁸², and R⁸³, each independently is H,        —CH₃, —CF₃, phenyl, benzyl, F, Cl, Br, I, —OCH₃, or —OH;    -   A⁵ is an optionally substituted Phe, an optionally substituted        1-Nal, or an optionally substituted 2-Nal;    -   A⁶ is Arg; and    -   A⁷ is Trp,

wherein any amino acid residue is either in L- or in D-configuration.

In example embodiments, A³ and A⁴, each independently, is a residue ofan amino acid selected from amino acids represented by the followingstructural formulas:

Values and preferred values of the remainder of the variables are asdefined herein with respect to Formula (I).

“Alkyl” used alone or as part of a larger moiety such as “hydroxyalkyl”,“alkoxyalkyl”, “alkylamine” refers to a straight or branched, saturatedaliphatic group having the specified number of carbons, typically having1 to 12 carbon atoms. More particularly, the aliphatic group may have 1to 8, 1 to 6, or 1 to 4 carbon atoms. This term is exemplified by groupssuch as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl,tert-butyl, n-hexyl, and the like.

“Haloalkyl” refers to an alkyl group substituted with one or morehalogen atoms.

“Halogen” and “halo” refer to fluoro, chloro, bromo or iodo.

“Cyano” refers to the group —CN.

“Ph” refers to a phenyl group.

“Carbonyl” refers to a divalent —C(O)— group.

“Aryl” used alone or as part of a larger moiety as in “aralkyl” refersto an aromatic carbocyclic group of from 6 to 18 carbon atoms having asingle ring or multiple condensed rings. The term “aryl” also includesaromatic carbocycle(s) fused to cycloalkyl or heterocycloalkyl groups.Examples of aryl groups include phenyl, benzo[d][1,3]dioxole, naphthyl,phenanthrenyl, and the like.

“Aryloxy” refers to an —OAr group, wherein O is an oxygen atom and Ar isan aryl group as defined above.

“Aralkyl” refers to an alkyl having at least one alkyl hydrogen atomreplaced with an aryl moiety, such as benzyl, —(CH₂)₂phenyl,—(CH₂)₃phenyl, —CH(phenyl)₂, and the like.

“Heteroaryl” used alone or a part of a larger moiety as in“heteroaralkyl” refers to a 5 to 18 membered monocyclic, bicyclic ortricyclic heteroaromatic ring system, containing one to four ringheteroatoms independently selected from nitrogen, oxygen and sulfur. Theterm “heteroaryl” also includes heteroaromatic ring(s) fused tocycloalkyl or heterocycloalkyl groups. Particular examples of heteroarylgroups include optionally substituted pyridyl, pyrrolyl, pyrimidinyl,furyl, thienyl, imidazolyl, oxazolyl, isoxazolyl, thiazolyl,isothiazolyl, pyrazolyl, 1,2,3-triazolyl, 1,2,4-triazolyl,1,2,3-oxadiazolyl, 1,2,4-oxadiazolyl, 1,2,5-oxadiazolyl,1,3,4-oxadiazolyl, 1,3,4-triazinyl, 1,2,3-triazinyl, benzofuryl,[2,3-dihydro]benzofuryl, isobenzofuryl, benzothienyl, benzotriazolyl,isobenzothienyl, indolyl, isoindolyl, 3H-indolyl, benzimidazolyl,imidazo[1,2-a]pyridyl, benzothiazolyl, benzoxa-zolyl, quinolizinyl,quinazolinyl, pthalazinyl, quinoxalinyl, cinnolinyl, napthyridinyl,pyrido[3,4-b]pyridyl, pyrido[3,2-b]pyridyl, pyrido[4,3-b]pyridyl,quinolyl, isoquinolyl, tetrazolyl, 1,2,3,4-tetrahydroquinolyl,1,2,3,4-tetrahydroisoquinolyl, purinyl, pteridinyl, carbazolyl,xanthenyl, benzoquinolyl, and the like.

“Heteroaralkyl” refers to an alkyl having at least one alkyl hydrogenatom replaced with a heteroaryl moiety, such as —CH₂-pyridinyl,—CH₂-pyrimidinyl, and the like.

“Alkoxy” refers to the group —O—R where R is “alkyl”, “cycloalkyl”,“alkenyl”, or “alkynyl”. Examples of alkoxy groups include for example,methoxy, ethoxy, ethenoxy, and the like.

“Hydroxyalkyl” and “alkoxyalkyl” are alkyl groups substituted withhydroxyl and alkoxy, respectively.

“Amino” means —NH₂; “alkylamine” and “dialkylamine” mean —NHR and —NR₂,respectively, wherein R is an alkyl group. “Cycloalkylamine” and“dicycloalkylamine” mean —NHR and —NR₂, respectively, wherein R is acycloalkyl group. “Cycloalkylalkylamine” means —NHR wherein R is acycloalkylalkyl group. “[Cycloalkylalkyl][alkyl]amine” means —N(R)₂wherein one R is cycloalkylalkyl and the other R is alkyl.

“Acyl” refers to R″—C(O)—, where R¹¹ is H, alkyl, substituted alkyl,heteroalkyl, substituted heteroalkyl, alkenyl, substituted alkenyl,aryl, alkylaryl, or substituted alklyaryl, and is indicated in thegeneral formula of a particular embodiment as “Ac”.

Suitable substituents for “alkyl”, “aryl”, or “heteroaryl”, etc., arethose which will form a stable compound of the invention. Examples ofsuitable substituents are those selected from the group consisting ofhalogen, —CN, —OH, —NH₂, (C1-C4)alkyl, (C1-C4)haloalkyl, aryl,heteroaryl, (C3-C7)cycloalkyl, (5-7 membered) heterocycloalkyl,—NH(C1-C6)alkyl, —N((C1-C6)alkyl)₂, (C1-C6)alkoxy,(C1-C6)alkoxycarbonyl, —CONH₂, —OCONH₂, —NHCONH₂, —N(C1-C6)alkylCONH₂,—N(C1-C6)alkylCONH(C1-C6)alkyl, —NHCONH(C1-C6)alkyl,—NHCON((C1-C6)alkyl)₂, —N(C1-C6)alkylCON((C1-C6)alkyl)₂, —NHC(S)NH₂,—N(C1-C6)alkylC(S)NH₂, —N(C1-C6)alkylC(S)NH(C1-C6)alkyl,—NHC(S)NH(C1-C6)alkyl, —NHC(S)N((C1-C6)alkyl)₂,—N(C1-C6)alkylC(S)N((C1-C6)alkyl)₂, —CONH(C1-C6)alkyl,—OCONH(C1-C6)alkyl —CON((C1-C6)alkyl)₂, —C(S)(C1-C6)alkyl,—S(O)_(p)(C1-C6)alkyl, —S(O)_(p)NH₂, —S(O)_(p)NH(C1-C6)alkyl,—S(O)_(p)N((C1-C6)alkyl)₂, —CO(C1-C6)alkyl, —OCO(C1-C6)alkyl,—C(O)O(C1-C6)alkyl, —OC(O)O(C1-C6)alkyl, —C(O)H or —CO₂H. Moreparticularly, the substituents are selected from halogen, —CN, —OH,—NH₂, (C1-C4)alkyl, (C1-C4)haloalkyl, (C1-C4)alkoxy, phenyl, and(C3-C7)cycloalkyl. Within the framework of this invention, said“substitution” is also meant to encompass situations where a hydrogenatom is replaced with a deuterium atom. p is an integer with a value of1 or 2.

Suitable substituents on a substituted Phe include one to fivesubstituents on any aromatic carbons, the substituents being selectedfrom F, Cl, Br, I, —CH₃, —OH, —CN, amine, —NO₂, or —OCH₃. Examplesinclude Phe(2′-F), Phe(2′-Cl), Phe(2′-Br), Phe(2′-I), Phe(2′-CN),Phe(2′-CH₃), Phe(2′-OCH₃), Phe(2′-CF₃), Phe(2′-NO₂), Phe(3′-F),Phe(3′-Cl), Phe(3′-Br), Phe(3′-I), Phe(3′-CN), Phe(3′-CH₃),Phe(3′-OCH₃), Phe(3′-CF₃), Phe(3′-NO₂), Phe(4′-F), Phe(4′-Cl),Phe(4′-Br), Phe(4′-I), Phe(4′-CN), Phe(4′-CH₃), Phe(4′-OCH₃),Phe(4′-CF₃), Phe(4′-NO₂), Phe(4′-t-Bu), Phe(2′,4′-diF), Phe(2′,4′-diCl),Phe(2′,4′-diBr), Phe(2′,4′-diI), Phe(2′,4′-di-CN), Phe(2′,4′-di-CH₃),Phe(2′,4′-di-OCH₃), Phe(3′,4′-diF), Phe(3′,4′-diCl), Phe(3′,4′-diBr),Phe(3′,4′-diI), Phe(3′,4′-di-CN), Phe(3′,4′-di-CH₃), Phe(3′,4′-di-OCH₃),Phe(3′,5′-diF), Phe(3′,5′-diCl), Phe(3′,5′-diBr), Phe(3′,5′-diI),Phe(3′, 5′-di-CN), Phe(3′,5′-diCH₃), Phe(3′,5′-di-OCH₃), orPhe(3′,4′,5′-triF).

Suitable substituents on a substituted His include one to threesubstituents on any substitutable ring atom, the substituents beingselected from F, Cl, Br, I, —CH₃, —OH, —CN, amine, —NO₂, benzyl, or—OCH₃. Examples include 1-Methyl-Histidine and 3-Methyl-Histidine.

Designation “(amino acid),” means that an amino acid is repeated ntimes. For example, designation “(Pro)₂” or “(Arg)₃” mean that prolineor arginine residues are repeated, respectively, two or three times.

Pharmaceutically acceptable salts of the compounds disclosed herein areincluded in the present invention. For example, an acid salt of acompound containing an amine or other basic group can be obtained byreacting the compound with a suitable organic or inorganic acid,resulting in pharmaceutically acceptable anionic salt forms. Examples ofanionic salts include the acetate, benzenesulfonate, benzoate,bicarbonate, bitartrate, bromide, calcium edetate, camsylate, carbonate,chloride, citrate, dihydrochloride, edetate, edisylate, estolate,esylate, fumarate, glyceptate, gluconate, glutamate,glycollylarsanilate, hexylresorcinate, hydrobromide, hydrochloride,hydroxynaphthoate, iodide, isethionate, lactate, lactobionate, malate,maleate, mandelate, mesylate, methylsulfate, mucate, napsylate, nitrate,pamoate, pantothenate, phosphate/diphospate, polygalacturonate,salicylate, stearate, subacetate, succinate, sulfate, tannate, tartrate,teoclate, tosylate, triethiodide, and trifluoroacetate salts.

Salts of the compounds containing an acidic functional group can beprepared by reacting with a suitable base. Such a pharmaceuticallyacceptable salt can be made with a base which affords a pharmaceuticallyacceptable cation, which includes alkali metal salts (especially sodiumand potassium), alkaline earth metal salts (especially calcium andmagnesium), aluminum salts and ammonium salts, as well as salts madefrom physiologically acceptable organic bases such as trimethylamine,triethylamine, morpholine, pyridine, piperidine, picoline,dicyclohexylamine, N,N′-dibenzylethylenediamine, 2-hydroxyethylamine,bis-(2-hydroxyethyl)amine, tri-(2-hydroxyethyl)amine, procaine,dibenzylpiperidine, dehydroabietylamine, N,N′-bisdehydroabietylamine,glucamine, N-methylglucamine, collidine, quinine, quinoline, and basicamino acids such as lysine and arginine.

Pharmaceutical Compositions

The invention further relates to a pharmaceutical composition comprisingan ionic complex comprising: a cationic polypeptide; an anionicexcipient selected from: a PEG-carboxylic acid; a fatty acid having 10or more carbon atoms; an anionic phospholipid; and a combinationthereof; and a pharmaceutically acceptable carrier. In one embodiment,the pharmaceutical composition further comprises an additional excipientas described below (e.g., carboxymethylcellulose (CMC)). For example,any of the anionic pospholipids described herein can be further combinedwith carboxymethyl cellulose (CMC), such as a combination ofmPEG-200-DSPE and CMC.

The ratio of concentrations of the cationic peptide and an anionicexcipient is determined in terms of molar ratio of a cationic charge inthe polypeptide to the charge of the anionic excipient. For example, interms of one positive charge in the polypeptide, the amount of theanionic excipient could range from 1:1 to 1:10. For additional positivecharges in the polypeptide, the ratio of anionic excipient may beadjusted accordingly. By varying the amount of anionic excipient withinthis ratio, the in vivo release characteristic of a polypeptide may bemodulated. A higher ratio typically can yield a composition thatprovides for a slower release of the polypeptide from the site ofadministration than the one with lower ratio.

The term “pharmaceutically acceptable carrier” in the pharmaceuticalcomposition of the invention, means a biocompatible, polar liquid. Thepolar nature of liquid assists in maintaining the complex in its ionicform. Biocompatible, polar liquids include, but are not limited to, aPEG (polyethylene glycol, e.g., a polyethylene glycol having an averagemolecular weight of 100 to 500), polyol (e.g., propylene glycol (PG),tripropylene glycol, glycerol), ethanol, benzyl alcohol, DMSO, NMP, DMF,water, pH buffered solutions, and mixtures thereof. It is understoodthat additional diluents and additional excipients as described belowcan be included in the pharmaceutical composition.

In certain embodiments, the pharmaceutical composition of the inventionforms a drug depot when injected into a subject. In some embodiments,the drug depot slowly releases the active compound over time followinginjection into a subject. In one aspect, at least a portion of thepharmaceutical composition precipitates to form a drug depot andreleases the pharmacologically active compound over time when injectedinto the subject. In some embodiments, the compositions of the inventionallow for high concentrations of polypeptide of structural Formula (I)appropriate for creating drug depots for sustained steady state releaseof therapeutically effective levels of the polypeptide in vivo. Anexample range of concentrations of polypeptide of structural Formula (I)in formulation is from about 0.001 mg/mL to about 100 mg/mL.

The pharmaceutical composition of the invention can include additionalexcipients (also referred to herein as co-excipients). Suitable examplesof additional excipients include pH stabilizing buffers, preservatives,surfactants, stabilizers, anti-oxidants, tonicity agents and ionic andnon-ionic polymers as defined herein.

Such a co-excipient can be added to aid in the formation of homogenoussuspensions and dispersion of the ionic complex or even the hydrophobicclass of anionic excipient, for example, a lipid or fatty acid. Theclass of co-excipients include the dispersing and emulsifying agentssuch as lecithin, soybean oil, castor oil, migliol, polyethylene glycol(MW ranging 200 to 5,000), methyl cellulose, and carboxymethylcellulose.

As used herein, the term “surfactant” refers to a surface-active agentor a substance that tends to reduce the surface tension of the liquid inwhich it is dissolved. Suitable surfactants include polysorbates,poloxamers, Triton, sodium dodecyl sulfate, sodium lauryl sulfate, andbetaines. For example, surfactants include polyoxyethylene (20) sorbitanmonolaurate (Tween® 20, e.g. from Sigma-Aldrich), polyoxyethylene (20)sorbitan monopalmitate (Tween® 40), polyoxyethylene (20) sorbitanmonooleate (Tween® 80), poloxamer 188, polyoxyethylene-polyoxypropyleneblock copolymer (Pluronic® F-68, e.g. from Sigma-Aldrich),polyethyleneglycol 660-12-hydroxystearate (Solutol® HS 15, BASF),cocamidopropyl betaine, linoleyl betaine, myristyl betaine, cetylbetaine, polyethoxylated castor oil (Cremophor®, now Kolliphor BASF),and lecithin.

As used herein, the term “tonicity agents” refers to substances used tomodulate the tonicity of a formulation. Tonicity in general relates tothe osmotic pressure of a solution usually relative to that of humanblood serum. The formulation can be hypotonic, isotonic or hypertonic. Aformulation is typically preferably isotonic. An isotonic formulation isliquid or liquid reconstituted from a solid form, e.g. from alyophilized form and denotes a solution having the same tonicity as someother solution with which it is compared, such as physiologic saltsolution and the blood serum. Tonicity agents can help to reduce painand irritation in a subject upon injection. Suitable tonicity agentsinclude dextrose, glycerin, hydroxyethyl starch, lactose, mannitol(e.g., D-mannitol), raffinose, sorbitol, sucrose, trehalose, sodiumchloride, calcium chloride, magnesium chloride, and potassium chloride.

The term “buffer” as used herein denotes an excipient which stabilizesthe pH of the pharmaceutical composition. Suitable buffers are wellknown in the art and can be found in the literature. Examples ofsuitable buffers include histidine-buffers, citrate-buffers,succinate-buffers, acetate-buffers and phosphate-buffers or mixturesthereof. Most preferred buffers comprise citrate, L-histidine ormixtures of L-histidine and L-histidine hydrochloride. Other preferredbuffer is acetate buffer. Independently from the buffer used, the pH canbe adjusted with an acid or a base known in the art, e.g. hydrochloricacid, acetic acid, phosphoric acid, sulfuric acid and citric acid,sodium hydroxide and potassium hydroxide.

The ionic polymers suitable for use as an additional excipient includeionic carboxymethyl cellulose (CMC), hyaluronic acid, poly(glutamicacid), poly(aspartic acid), poly(glutamic acid-co-glycine),poly(aspartic acid-co-glycine), poly(glutamic acid-co-alanine),poly(aspartic acid-co-alanine), starch glycolate, polygalacturonic acid,poly(acrylic acid), carrageenan and alginic acid.

In certain embodiments, the CMC has an average molecular weight range ofabout 5,000 to about 700,000. In some embodiments, methyl cellulose andcarboxymethyl cellulose may help modulate the viscosity of the ioniccomplex to enhance a slow releasing drug depot of the active compoundand release the active compound over time when injected into a subject.

The non-ionic polymers suitable for use as an additional excipientinclude polyethylene glycol having a MW ranging from about 100 to100,000, for example from about 100 to about 60,000, such as from about100 to about 10,000 such as from about 100 to about 5000. Other weightranges include from about 200 to about 60,000. Suitable examples ofneutral polymers include PEG-3350 and PEG-3400. The polyethylene glycolpolymers include both PEG and mPEG, which can be either monomethoxy ordimethoxy. PEG can be structurally depicted as follows:

HO—(CH₂CH₂O)_(n)—CH₂CH₂—OH

with the value of “n” varying with molecular weight. For example, thePEG-3500 acid has a molecular weight of 3,500.

The mPEG can have on of the following two structures:

Monomethoxy mPEG: CH₃O—(CH₂CH₂O)_(n)—CH₂CH₂—OH. One end capped asmethoxy group and the other end has a hydroxyl (OH) group.Di-methoxy mPEG: CH₃O—(CH₂CH₂O)_(n)—CH₂CH₂—OCH₃. Both the ends arecapped as methoxy groups

Diluents can be added to pharmaceutical compositions of the invention tofurther dissolve or suspend the ionic complex of the invention. Diluentsinclude biologically compatible materials such as non aqueous injectableliquids of low viscosity. Non aqueous injectable liquids of lowviscosity include castor oil, vegetable oil, mineral oil, squalene,monoglycerides, diglycerides, triglycerides, or mixtures of glycerides.In some embodiments, the diluent is Miglyol® 812 (from Sasol GmbH,Germany), Labrafac® WL1349 (caprylic acid triglyceride from Gattefossecompany, France), or Lipoid MCT (from Lipoid company, Germany) and thelike.

As used herein, a “drug depot” refers to the precipitate that can formfollowing injection of the pharmaceutical composition of the invention.The compositions can form a slow releasing drug depot of the activecompound and release the active compound over time when injected into asubject. In one aspect, at least a portion of the compositionprecipitates and releases the pharmacologically active compound overtime when injected into the subject.

A “preservative” is a compound which can be added to the formulation toreduce bacterial activity or undesirable chemical changes in theformulations. Examples of preservatives include benzyl alcohol, ethanol,methanol, isopropanol, butyl paraben, ethyl paraben, methyl paraben,propyl paraben, cathechol, 2-chlorophenol, m-cresol, phenol, resorcinol,xylitol, 2,6-dimethylcyclohexanol, 2-methyl-2,4-pentadiol,polyvinylpyrrolidone, benzethonium chloride, merthiolate (thimersosal),benzoic acid, benzalkonium chloride, chlorobutanol, sodium benzoate,sodium propionate, and cetylpyridinium chloride.

Methods of Treatment

The invention relates to the use of the the ionic complex andpharmaceuctical composition comprising same to treat a subject sufferingfrom a disease or disorder that is responsive to the pharmacologicalactivity possessed by the cationic polypeptide of the ionic complex. Inone embodiment, the disorder to be treated is responsive to themodulation of MC4R in a subject in need of treatment. The methodcomprises administering to the subject an effective amount of an ioniccomplex comprising as the cationic polypeptide an MC4R modulator such asthose described in Formula I herein. In a particular embodiment, thedisorder responsive to modulation of the MC4R includes type 1 diabetes,type 2 diabetes, obesity, insulin resistance, metabolic syndrome, maleerectile dysfunction, female sexual disorder, non-alcoholic fatty liverdisease, non-alcoholic steatohepatitis, disorders of substance abuse,including alcoholism feeding disorders, cachexia, inflammation andanxiety.

In certain embodiments, the ionic complex and pharmaceutical compositioncomprising a cationic polypeptide of Formula I can possess higherselectivity and potency for the MC4R and melanocortin-3 receptor (MC3R)when compared to melanocortin-1 receptor (MC1R). The ionic complex andpharmaceutical composition of the present invention can reduce oreliminate such undesirable side effects as increase in blood pressureeffects, increase in heart rate, undesired effects on sexual arousal,and increase in skin pigmentation.

As used herein, the phrase “a disorder responsive to the modulation ofthe melanocortin-4 receptor” refers to any disorder that can be treatedby activation (agonizing) or inhibition of MC4R. Examples of suchdisorders will be described in detail below.

As used herein, the term “modulator” refers to compounds which interactwith the target receptor and affects its biological function. Examplesof modulators include full agonists, partial agonists, neutralantagonists, and inverse agonists.

As used herein, the term “agonist” refers to any chemical compound,either naturally occurring or synthetic, that, upon interacting with(e.g., binding to) its target, here, MC4R, raises the signaling activityof MC4R above its basal level. An agonist can be a superagonist (i.e. acompound that is capable of producing a greater maximal response thanthe endogenous agonist for the target receptor, and thus has an efficacyof more than 100%), a full agonist (i.e. a compound that elicits amaximal response following receptor occupation and activation) or apartial agonist (i.e. a compounds that can activate receptors but areunable to elicit the maximal response of the receptor system). Examplesof MC4R agonists will be described in detail below.

As used herein, the term “antagonist” refers to any chemical compound,that, upon interacting with (e.g., binding to) its target, here, MC4R,blocks, in a dose dependent manner, the signaling activity of an agonistcompound with the MC4R.

As used herein, the term “inverse agonist” refers to any chemicalcompound, that, upon interacting with (e.g., binding to) its target,here, MC4R, decreases, in a dose dependent manner, the basal level ofsignaling activity of the MC4R.

As used herein, an “effective amount” refers to an amount of apharmacologically active agent either as the ionic complex or a apharmaceutical composition comprising the ionic complex that istherapeutically or prophylactically sufficient to treat the targetdisorder. Examples of effective amounts typically range from about0.0001 mg/kg of body weight to about 500 mg/kg of body weight. Anexample range is from about 0.001 mg/kg of body weight to about 500mg/kg. For example, the effective amount can range from about 0.005mg/kg to about 500 mg/kg. In other examples, the range can be from about0.0001 mg/kg to about 5 mg/kg. In still other examples, effectiveamounts range from about 0.01 mg/kg of body weight to 50 mg/kg of bodyweight, or from 0.01 mg/kg of body weight to 20 mg/kg of body weight.

As used herein, the term “second agent” includes any activepharmaceutical ingredient (API) that, in combination with a peptidedescribed herein, enhances the therapeutic effect produced by a peptidedescribed herein alone or shows synergy with a peptide described herein(i.e. shows the combined effect that is greater than the additiveeffect). As used herein, “an enhanced therapeutic effect” includes animproved therapeutic profile other than synergy. Examples of enhancedtherapeutic effects include lowered effective dose of a peptidedescribed herein, prolonged therapeutic window of a peptide describedherein, etc. One or more second agents can be administered. Examples ofsecond agents will be described in detail below.

A second agent can be administered before, simultaneously with, or afterthe administration of a peptide described herein. Accordingly, a peptidedescribed herein and a second agent can be administered together in asingle formulation or can be administered in separate formulations,e.g., either simultaneously or sequentially. For example, if a peptidedescribed herein and a second agent are administered sequentially inseparate compositions, a peptide described herein can be administeredbefore or after a second therapeutic agent. In addition, a peptidedescribed herein and a second agent may or may not be administered onsimilar dosing schedules. For example, a peptide described herein and asecond therapeutic agent may have different half-lives and/or act ondifferent time-scales such that a peptide described herein isadministered with greater frequency than the second therapeutic agent orvice-versa. Finally, a peptide described herein can be followed by asecond agent, which further enhances therapeutic efficacy, as a resultof the consecutive application of both therapeutic agents. Either apeptide described herein or a second agent can be administered acutelyor chronically.

An effective amount can be achieved in the methods or compositions ofthe invention by co-administering a first amount of a compound having anMC4R modulator activity or a pharmaceutically acceptable salt thereofand a second amount of at least one second agent. In one embodiment, apeptide described herein and second agent are each administered in arespective effective amount (i.e., each in an amount which would betherapeutically effective if administered alone). In another embodiment,a peptide described herein and a second agent are each administered inan amount which alone does not provide a therapeutic effect (asub-therapeutic dose). In yet another embodiment, a peptide describedherein can be administered in an effective amount, while the secondagent is administered in a sub-therapeutic dose. In still anotherembodiment, a peptide described herein can be administered in asub-therapeutic dose, while the second agent is administered in aneffective amount. In example embodiment, a combination of a peptidedescribed herein and a second agent exhibits enhanced therapeutic effector synergy compared to either a peptide described herein or a secondagent alone.

The presence of a synergistic effect can be determined using suitablemethods for assessing drug interaction. Suitable methods include, forexample, the Sigmoid-Emax equation (Holford, N. H. G. and Scheiner, L.B., Clin. Pharmacokinet. 6: 429-453 (1981)), the equation of Loeweadditivity (Loewe, S. and Muischnek, H., Arch. Exp. Pathol. Pharmacol.114: 313-326 (1926)), and the median-effect equation (Chou, T. C. andTalalay, P., Adv. Enzyme Regul. 22: 27-55 (1984)). Each equationreferred to above can be applied with experimental data to generate acorresponding graph to aid in assessing the effects of the drugcombination. The corresponding graphs associated with the equationsreferred to above are the concentration-effect curve, isobologram curveand combination index curve, respectively.

As used herein, the term “subject” refers to a mammal, preferably ahuman, but can also mean an animal in need of veterinary treatment,e.g., companion animals (e.g., dogs, cats, and the like), farm animals(e.g., cows, sheep, pigs, horses, and the like) and laboratory animals(e.g., rats, mice, guinea pigs, and the like).

As used herein “treating” includes achieving, partially orsubstantially, delaying, inhibiting or preventing the progression ofclinical indications related to the target disorder. For example,“treating” includes achieving, partially or substantially, one or moreof the following results: partially or totally reducing the body weight(as measured, for example, by a body mass index, BMI); ameliorating orimproving a clinical symptom or indicators associated with obesity, suchas type-2 diabetes, pre-diabetic condition, blood level of hemoglobinA1C (Hb1Ac) above 6%, hyperinsulimenia, hyperlipidemia, insulininsensitivity, glucose intolerance etc; delaying, inhibiting orpreventing the progression of obesity and obesity related indication; orpartially or totally delaying, inhibiting or preventing the onset ordevelopment of obesity or obesity related indication. Delaying,inhibiting or preventing the progression of the obesity includes forexample, delaying, inhibiting or preventing the progression of a subjecthaving normal weight to obesity. The term “treating” further includespartially or totally reducing the risk for coronary artery disease,stroke, and diabetes (e.g. type 2) associated with the metabolicsyndrome as well as ameliorating or improving a clinical symptom orsigns of metabolic syndrome associated with metabolic syndrome, such asany one or more of the five indicators listed above. For example, theterm “treating” includes delaying, inhibiting or preventing theprogression of parameters associated with the metabolic syndrome,including insulin resistance, glucose clearance and parameters ofcardiovascular disease including heart rate and blood pressure, jointdisease, inflammation, sleep apnea, binge eating and other eatingdisorders including bulimia, supportive therapy for weight loss surgeryand supportive weight loss therapy prior to orthopedic surgery.“Prophylactic treatment” refers to treatment before onset of clinicalsymptoms of a target disorder to prevent, inhibit or reduce itsoccurrence.

Responsive Disorders

Examples of disorders responsive to the modulation of MC4R and moregenerally disorders responsive to the pharmacological action of thepolypeptide categories and specific polypeptide listed above, includeacute and chronic inflammatory diseases such as general inflammation,inflammatory bowel disease, brain inflammation, sepsis and septic shock;diseases with an autoimmune component such as rheumatoid arthritis,gouty arthritis, and multiple sclerosis; metabolic diseases and medicalconditions accompanied by weight gain such as obesity, feeding disordersand Prader-Willi Syndrome; metabolic diseases and medical conditionsaccompanied by weight loss such as anorexia, bulimia, AIDS wasting,cachexia, cancer cachexia and wasting in frail elderly; diabetes anddiabetalogical related conditions and complications of diabetes such asretinopathy; neoplastic proliferation such as skin cancer, and prostatecancer; reproductive or sexual medical conditions such as endometriosisand uterine bleeding in women, sexual dysfunction, erectile dysfunctionand decreased sexual response in females; diseases or conditionsresulting from treatment or insult to the organism such as organtransplant rejection, ischemia and reperfusion injury, treatment ofspinal cord injury and to accelerate wound healing, as well as weightloss caused by chemotherapy, radiation therapy, temporary or permanentimmobilization or dialysis; cardiovascular diseases or conditions suchas hemorrhagic shock, cardiogenic shock, hypovolemic shock,cardiovascular disorders and cardiac cachexia; pulmonary diseases orconditions such as acute respiratory distress syndrome, chronicobstructive pulmonary disease, asthma and pulmonary fibrosis; to enhanceimmune tolerance and to combat assaults to the immune system such asthose associated with certain allergies or organ transplant rejection;treatment of dermatological diseases and conditions such as psoriasis,skin pigmentation depletion, acne, keloid formation and skin cancer;behavioral, central nervous system or neuronal conditions and disorderssuch as anxiety, depression, memory and memory dysfunction, modulatingpain perception and treating neuropathic pain; conditions and diseasesassociated with alcohol consumption, alcohol abuse and/or alcoholism;and renal conditions or diseases such as the treatment of renal cachexiaor natriuresis. Additional examples include normalizing or homeostaticactivities in a subject, including thyroxin release, aldosteronesynthesis and release, body temperature, blood pressure, heart rate,vascular tone, brain blood flow, blood glucose levels, bone metabolism,bone formation or development, ovarian weight, placental development,prolactin and FSH secretion, intrauterine fetal growth, parturition,spermatogenesis, sebum and pheromone secretion, neuroprotection andnerve growth as well as modulating motivation, learning and otherbehaviors. Further examples include binge eating, bulimia or othereating disorders.

In example embodiments, the disorders responsive to the modulation ofthe MC4R receptor are type 1 diabetes, type 2 diabetes, obesity, insulinresistance, metabolic syndrome, cardiovascular disease, or low densitylipoprotein/high density lipoprotein/triglyceride imbalance,non-alcoholic fatty liver disease, and disorders of substance abuse.

In example embodiments, the disorders responsive to the modulation ofthe MC4R receptor is type 1 diabetes, type 2 diabetes, obesity, insulinresistance or metabolic syndrome.

Obesity

As used herein, the term “obese” refers to a subject having a body massindex (BMI) of about 30 kg/m² or higher, e.g., a BMI of 25, 26, 27, 28,29, 30, 31, 32, 33, 34, 35, 36, 37 kg/m², or more. In particularembodiments, an obese subject has a BMI within the ranges defined as“obese” by the Center for Desease Control. See, URLhttp://www.cdc.gov/obesity/defining.html, last accessed on Oct. 28,2011. For example, in some embodiments, an adult who has a BMI >=30.0kg/m² is obese.

Diabetes and Related Disorders

In example embodiments, subjects to be treated by the methods providedby the invention have or are at increased risk for developing diabetesrelated disorders. “Diabetes-related disorders,” refers to diabetes(including type 1 (OMIM 222100) and type 2 (OMIM 125853)), insulinresistance and metabolic syndrome.

In example embodiments, the subject to be treated has diabetes (type 1or type 2), insulin resistance or metabolic syndrome. In exampleembodiments, the disorder is diabetes, e.g. type 2 diabetes. In exampleembodiments, the subject has, or is at increased risk for developing,type 2 diabetes as defined by the World Health Organization and theInternational Diabetes Federation in “Definition and diagnosis ofdiabetes mellitus and intermediate hyperglycaemia,” published in 2006,which is incorporated by reference in its entirety. In exampleembodiments, a diabetic subject exhibits a fasting plasma glucoseof >=126 mg/dL or a 2-hour plasma glucose (2 hours after oraladministration of 75 glucose) >=200 mg/dL. In example embodiments adiabetic or pre-diabetic subject exhibits elevated levels of glycatedhemoglobin, e.g., greater than 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2,5.3, 5.4, 5.5, 5.6, 5.8, 6.0, 6.2, 6.4, 6.6, 6.8, 7.0, 7.2, 7.4, 7.6%,or more of total hemoglobin. In example embodiments, a diabetic orpre-diabetic subject may be identified or further characterized bygenetic polymorphisms (including, for example, polymorphisms leading toaltered expression levels, e.g., elevated or reduced expression levelsand/or variations in coding sequences) in or near one or more of thegenes in Table 1, below:

TABLE 1 Gene/ OMIM Location Locus No. 2q24.1 GPD2 138430 2q31.3 NEUROD1601724 2q36.3 IRS1 147545 3p25.2 PPARG 601487 3q27.2 IGF2BP2 6082894p16.1 WFS1 606201 5q34-q35.2 NIDDM4 608036 6p22.3 CDKAL1 611259 6p21.31HMGA1 600701 6q23.2 ENPP1 173335 7p13 GCK 138079 7q32.1 PAX4 1674138q24.11 SLC30A8 611145 10q25.2-q25.3 TCF7L2 602228 11p15.1 KCNJ11 60093711p15.1 ABCC8 600509 11p11.2 MAPK8IP1 604641 12q24.31 HNF1A 14241013q12.2 IPF1 600733 13q34 IRS2 600797 15q21.3 LIPC 151670 17p13.1 SLC2A4138190 17q12 HNF1B 189907 17q25.3 GCGR 138033 19p13.2 RETN 60556519p13.2 RETN 605565 19q13.2 AKT2 164731 20q12-q13.1 NIDDM3 60369420q13.12 HNF4A 600281 20q13.13 PTPN1 176885

In example embodiments, additional genes that can be used to identify orfurther characterize subjects to be treated by the methods provided bythe invention include FTO (OMIM 610966), JAZF1 (OMIM 606246) and HHEX604420).

In example embodiments, a subject to be treated by the methods providedby the invention has type 1 diabetes. In example embodiments, a subjectwith type 1 diabetes is characterized by virtue of a C-peptide assay,e.g., fasting C-peptide levels of less than about 1.0 nmol/L, e.g., lessthan 1.2, 1.1, 1.0, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4 nmol/L, or less, e.g.,less than 0.33, 0.25, 0.2, or 0.1 nmol/L. In example embodiments,C-peptide levels are measured after oral glucose challenge (2 hoursafter oral administration of 75 g of glucose) and an increase of lessthan 0.54 nmol/L, e.g., less than 0.50, 0.45, 0.40, 0.35, 0.30, 0.25,0.20, 0.15, or 0.10 nmol/L is detected. Impaired fasting glucose(110-125 mg/dL) or impaired glucose tolerance (2 hours after oraladministration of 75 g of glucose challenge: 140-199 mg/dL) may be usedto identify or further characterize the reduced beta-cell function insubjects with type 1 diabetes. In example embodiments, type1 diabeticsare identified or further characterized by the presence ofautoantibodies to islet cell antigens and/or insulin, e.g.,autoantibodies directed to 65 kDa of GAD (OMIM 138275) and/orphosphatase-related IA-2 molecule.

Insulin Resistance

In example embodiments, the disorder is “insulin resistance,” which maybe identified by any means known in the art, and is characterized by areduced ability of insulin to lower blood glucose levels. In exampleembodiments, the insulin resistance is identified or furthercharacterized by the presence of one or more polymorphisms (including,for example, polymorphisms leading to altered expression levels, e.g.,elevated or reduced expression levels as well as coding sequencevariants of gene products, such as proteins) in one or more of thefollowing genes: RETN, PTPN1, TCF1 (OMIM 142410; see, e.g., polymorphism0011), PPP1R3A (OMIM 600917; see, e.g., polymorphisms 0001, 0003), PTPN1176885; see, e.g., polymorphism 0001), ENPP1 (OMIM 173335; see, e.g.,polymorphism 0006), IRS1 (OMIM 147545; see, e.g., polymorphism 0002),EPHX2 (OMIM 132811; see, e.g., polymorphism 0001), leptin (OMIM 164160,see, e.g., polymorphisms 0001 and 0002), leptin receptor (OMIM 601007,see, e.g., polymorphisms 0001, 0002, 0004, and 0005), or the insulinreceptor (INSR, OMIM 147670, see, e.g., polymorphisms 0001-0037).

Metabolic Syndrome

In example embodiments, the disorder is metabolic syndrome. As usedherein, the term “metabolic syndrome” refers to a group of symptoms thatoccur together and increase the risk for coronary artery disease,stroke, and type 2 diabetes. According to the American Heart Associationand the National Heart, Lung, and Blood Institute, metabolic syndromealso referred to as Syndrome X) is present if a subject has three ormore of the following signs:

1) Blood pressure equal to or higher than 130/85 mmHg;

2) Fasting blood sugar (glucose) equal to or higher than 100 mg/dL;

3) Large waist circumference (length around the waist):

-   -   Men—40 inches or more;    -   Women—35 inches or more;

4) Low HDL cholesterol:

-   -   Men—under 40 mg/dL;    -   Women—under 50 mg/dL;

5) Triglycerides equal to or higher than 150 mg/dL.

Metabolic syndrome can be diagnosed by testing the subject's bloodpressure, blood glucose level, HDL cholesterol level, LDL cholesterollevel, total cholesterol level, and triglyceride level.

In example embodiments the subject has central obesity (waistcircumference >=80 cm for women; >=90 cm for Asian men, including ethnicSouth and Central Americans, and >=94 cm for all other males), BMI>30kg/m², raised triglycerides (>=150 mg/dL, or specific treatment for thislipid abnormality), reduced HDL cholesterol (<40 mg/dL in males, <50mg/dL in females or specific treatment for this lipid abnormality),raised blood pressure (sBP>=130 mmHg or dBP>=85 mmHg or treatment ofpreviously diagnosed hypertension) or raised fasting plasma glucose(FPG>=100 mg/dL or previous type 2 diabetes diagnosis), includingcombinations thereof. In example embodiments, the subject to be treatedby the methods provided by the invention has or is at increased risk formetabolic syndrome, as defined by the International Diabetes Federationin “The IDF consensus worldwide definition of the metabolic syndrome,”published in 2006, which is incorporated by reference in its entirety,i.e., the subject has central obesity (as described above, and/or BMI>30kg/m²) AND any two of raised triglyceries, reduced HDL cholesterol,raised blood pressure, or raised fasting plasma glucose. In exampleembodiments, metabolic syndrome is characterized, or furthercharacterized, by the presence of a mutation at a locus selected from3q27 (see, for example, OMIM 605552) and/or 17p12 (see, for example,OMIM 605572) in the subject.

Disorders Caused by MC4R Mutations

The present invention relates to a method of treating a disorder in asubject suffering from an attenuated response of MC4R to α-melanocortinstimulating hormone (α-MSH). The method comprises administering aneffective amount of an agonist of the melanocortin-4 receptor (MC4R). Inan example embodiment, the subject is a heterozygous carrier of an MC4Rmutation resulting in the attenuated response of MC4R to α-melanocortinstimulating hormone (α-MSH). Because heterozygous carriers retain anability to respond to the natural ligand of MC4R, treatingMC4R-associated disorders in heterozygous carriers by administration ofan MC4R agonist does not rely on the knowledge of the type of the MC4Rmutation.

In one example embodiment, the disorder is obesity, for example,MC4R-associated obesity. In another example embodiment, the disorder ismetabolic syndrome.

Human MC4R gene (hMC4R) is a well-characterized protein encoded by agenomic sequence having GenBank accession number CH471077.

Mutations in the MC4R receptor are an associated cause of severechildhood obesity. The carrier prevalence for MC4R mutations in ajuvenile-onset obese population has been noted to be around 2.5% with ahighest prevalence of 6% among severe obese children. Humans with MC4Rmutations show a more or less similar phenotype as has been describedfor mice with mutations in the MC4 receptor gene. Those people showclear hyperphagia, hyperinsulinaemia, increased fat mass, accompanied bylean body mass, bone mineral density and linear growth rate, with nochanges in cortisol levels, gonadotropin, thyroid and sex steroidlevels. In contrast to MC4 receptor deletion, hyperphagia andhyperinsulinaemia tends to subside with age in human subjects. Similarto the MC4R knockout mice, the phenotype in heterozygote carriers isintermediate in comparison to homozygote carriers. The exhibitedhyperphagia observed upon a test meal is less severe than that observedin people with a leptin deficiency. The severity of MC4 receptordysfunction seen in assays in vitro can predict the amount of foodingested at a test meal by the subject harboring that particularmutation and correlates with the onset and severity of the obesephenotype. At least 90 different MC4 receptor mutations have beenassociated with obesity and additional mutations in the MC4 receptor arelikely to be discovered, leading to a similar obesity phenotype.

Examples of the MC4R mutations that cause obesity in humans aredescribed in Farooqi et al., The Journal of Clinical Investigation, July2000, vol. 106 (2), pp. 271-279 and Vaisse et al., The Journal ofClinical Investigation, July 2000, vol. 106(2), pp. 253-262, therelevant portions of which are incorporated herein by reference.

Additional mutations that potentially cause obesity in humans include,R18H, R18L, S36Y, P48S, V50M, F51L, E61K, I69T, D90N, S94R, G98R, I121T,A154D, Y157S, W174C, G181D, F202L, A219 V, I226T, G231S, G238D, N240S,C271R, S295P, P299L, E308K, I317V, L325F, and 750DelGA, as described inXiang et al., “Pharmacological characterization of 30 humanmelanocortin-4 receptor polymorphisms with the endogenousproopiomelanocortin-derived agonists, synthetic agonists, and theendogenous agouti-related protein antagonist.” Biochemistry, 2010 Jun.8; 49(22):4583-600, the relevant portions of which are incorporatedherein by reference.

Further examples of mutations that potentially cause obesity in humansare those listed in Online Mendelian Inheritance in Man (OMIM), adatabase of human genes and genetic disorders, under the accessionnumber 155541 (MC4R) (more precisely, accession nos.155541.0001-155541.0023) at the URL http://omim.org/entry/155541.Representative examples include 4-BP DEL, NT631; 4-BP INS, NT732;TYR35TER; ASP37VAL; SER58CYS; ILE102SER; ASN274SER; 1-BP INS, 112A; 4-BPDEL, 211CTCT; ILE125LYS; ALA175THR; ILE316SER; TYR287TER; ASN97ASP;15-BP DEL (delta88-92 codons); and SER127LEU. The relevant portions ofthe OMIM database are incorporated herein by reference.

In example embodiments, the MC4R mutation results in retention of theMC4R signaling activity.

Mutations in the genomic sequence encoding MC4R can be detected by themethods that are well known to a person of ordinary skill in the art.For example, the genomic sequence can be cloned using nucleotideprimers, such as e.g., the primers described in Farooqi et al., TheJournal of Clinical Investigation, July 2000, vol. 106 (2), pp. 271-279and Vaisse et al., The Journal of Clinical Investigation, July 2000,vol. 106(2), pp. 253-262, and the cloned sequence analyzed usingcommercially available sequencers and software.

Activity of MC4R can be measured by the methods well known to a personof ordinary skill in the art. For example, cells can be transientlytransfected with the cloned MC4R DNA, the transfected cells contacted byan agonist of MC4R (e.g. α-MSH), and the intracellular level of cAMP,the secondary messenger of MC4R, measured by an electrochemiluminescenceassay described, e.g., in Roubert et al., Journal of Endocrinology(2010) 207, pp. 177-183. A reduction in MC4R signaling can beascertained by comparing the intracellular level of cAMP produced inresponse to a given agonist by a wild type MC4R to that produced by amutant MC4R.

MC4R modulators (e.g. agonists) may also be used to treat patientssuffering from other disorders, such as reduced tone of the naturalagonists of the MC4R. Example of such patients include individualsheterozygous or homozygous for mutations in the genes important inleptin-dependent pathway (Nature Clinical Practice Endocrinology andMetabolism, 2006; 2; 6; 318 and NEng J Med: 2007; 356; 3; 237),proopiomelanocortin processing (Nature Genetics, 1998, 155; CellMetabolism, 2006; 3; 135; Annals Acad Med, 2009, 38; 1; 34), ormutations in the genes coding for prohormone convertases.

Modes of Administration

Administration of a ionic complex or pharmaceutical compositiondescribed herein useful to practice the methods described herein, can becontinuous, hourly, four times daily, three time daily, twice daily,once daily, once every other day, twice weekly, once weekly, once everytwo weeks, once a month, or once every two months, once every 3 months,once every 4 months, once every 5 months or once every 6 months orlonger or some other intermittent dosing regime. The ionic complecompositions of this invention are suitable for effective therapeuticadministration ranging from once daily, once weekly, once every 2 weeks,once every four weeks, once every 2 months, once every 3 months, onceevery 4 months, once every 5 months or once every 6 months.

Suitable methods of administration include, but are not limited toperipheral administration. Examples of peripheral administration includeoral, subcutaneous, intraperitoneal, intramuscular, intravenous, rectal,transdermal, buccal, sublingual, inhalation, pulmonary, or intranasalforms of administration. Preferred embodiments use subcutaneousadministration.

Combination Therapy

Any peptide described herein whether part of an ionic complex oruncomplexed (e.g., Peptide 1 before complexation) can be used fortreatment of any of the disorders responsive to the modulation of MC4R,by administration in combination with one or more other pharmaceuticallyactive compounds (“second agent”). Such combination administration canbe by means of a single dosage form which includes one or more peptidesdescribed herein and one or more second agents, such single dosage formsinclude a tablet, capsule, spray, inhalation powder, injectable liquid,or the like. Alternatively, combination administration can be by meansof administration of two different dosage forms, with one dosage formcontaining one or more peptides described herein, and the other dosageform including one or more second agents. In this instance, the dosageforms may be the same or different. Without meaning to limit combinationtherapies, the following exemplifies certain combination therapies whichmay be employed.

A peptide described herein (e.g., as part of the ionic complex oruncomplexed) can be combined with one or more second agents useful inthe treatment of various weight and feeding-related disorders, such asobesity and/or overweight. In particular, a second agent can be ananti-obesity drug that affects energy expenditure, glycolysis,gluconeogenesis, glucogenolysis, lipolysis, lipogenesis, fat absorption,fat storage, fat excretion, hunger and/or satiety and/or cravingmechanisms, appetite/motivation, food intake, or gastrointestinalmotility. Drugs that reduce energy intake include, in part, variouspharmacological agents, referred to as anorectic drugs, which are usedas adjuncts to behavioral therapy in weight reduction programs.

Generally, a total dosage of the obesity control agents or medications,when used in combination with one or more peptide described herein canrange from 0.1 to 3,000 mg/day, preferably from about 1 to 1,000 mg/day,and more preferably from about 1 to 200 mg/day in single or 2-4 divideddoses. The exact dose, however, is determined by the attending clinicianand is dependent on such factors as the potency of the compoundadministered, the age, weight, condition, and response of the patient.

One or more peptides described herein (either as part of an ioniccomplex or uncomplexed) can be combined with one or more second agentsuseful in the treatment of diabetes.

One or more peptides described herein can in addition or alternativelyfurther be combined with one or more second agents useful in thetreatment of diseases, disorders and/or conditions associated withobesity and/or overweight, such as insulin resistance; impaired glucosetolerance; type 2 diabetes; metabolic syndrome; dyslipidemia (includinghyperlipidemia); hypertension; heart disorders (e.g. coronary heartdisease, myocardial infarction); cardiovascular disorders; non-alcoholicfatty liver disease (including non-alcoholic steatohepatitis); jointdisorders (including secondary osteoarthritis); gastroesophageal reflux;sleep apnea; atherosclerosis; stroke; macro and micro vascular diseases;steatosis (e.g. in the liver); gall stones; and gallbladder disorders.

Second Agent

The one or more second agents are, for example, selected from:

-   -   insulin and insulin analogues;        -   insulin secretagogues, including sulphonylureas (e.g.            glipizide) and prandial glucose regulators (sometimes called            “short-acting secretagogues”), such as meglitinides (e.g.            repaglinide and nateglinide);    -   agents that improve incretin action: an incretin, an incretin        mimetic, an agents that improves incretin function e.g. GLP-1,        GIP; GLP-1 agonists (e.g., exenatide and liraglutide (VICTOZA)),        DPP-4 inhibitors (e.g. vildagliptin, saxagliptin, and        sitagliptin)        -   insulin sensitising agents including peroxisome proliferator            activated receptor gamma (PPARγ) agonists, such as            thiazolidinediones (e.g. pioglitazone and rosiglitazone),            and agents with any combination of PPAR alpha, gamma and            delta activity;        -   agents that modulate hepatic glucose balance, for example            biguanides (e.g. metformin), fructose 1,6-bisphosphatase            inhibitors, glycogen phopsphorylase inhibitors, glycogen            synthase kinase inhibitors, and glucokinase activators;        -   agents designed to reduce/slow the absorption of glucose            from the intestine, such as alpha-glucosidase inhibitors            (e.g. miglitol and acarbose);        -   agents which antagonize the actions of or reduce secretion            of glucagon, such as amylin analogues (e.g. pramlintide);        -   agents that prevent the reabsorption of glucose by the            kidney, such as sodium-dependent glucose transporter 2            (SGLT-2) inhibitors (e.g. dapagliflozin);        -   agents designed to treat the complications of prolonged            hyperglycaemia, such as aldose reductase inhibitors (e.g.            epalrestat and ranirestat);        -   agents used to treat complications related to            micro-angiopathies;        -   anti-dyslipidemia agents, such as HMG-CoA reductase            inhibitors (statins, e.g. rosuvastatin) and other            cholesterol-lowering agents;        -   PPARα agonists (fibrates, e.g. gemfibrozil and fenofibrate);        -   bile acid sequestrants (e.g. cholestyramine);        -   cholesterol absorption inhibitors (e.g. plant sterols (i.e.            phytosterols), synthetic inhibitors);        -   cholesteryl ester transfer protein (CETP) inhibitors;            inhibitors of the ileal bile acid transport system (IBAT            inhibitors);        -   bile acid binding resins;        -   nicotinic acid (niacin) and analogues thereof;        -   anti-oxidants, such as probucol;        -   omega-3 fatty acids;        -   antihypertensive agents, including adrenergic receptor            antagonists, such as beta blockers (e.g. atenolol), alpha            blockers (e.g. doxazosin), and mixed alpha/beta blockers            (e.g. labetalol);        -   adrenergic receptor agonists, including alpha-2 agonists            (e.g. clonidine);        -   angiotensin converting enzyme (ACE) inhibitors (e.g.            lisinopril), calcium channel blockers, such as            dihydropyridines (e.g. nifedipine), phenylalkylamines (e.g.            verapamil), and benzothiazepines (e.g. diltiazem);        -   angiotensin II receptor antagonists (e.g. candesartan);            aldosterone receptor antagonists (e.g. eplerenone);        -   centrally acting adrenergic drugs, such as central alpha            agonists (e.g. clonidine); and diuretic agents (e.g.            furosemide);        -   haemostasis modulators, including antithrombotics, such as            activators of fibrinolysis;        -   thrombin antagonists;        -   factor VIIa inhibitors; anticoagulants, such as vitamin K            antagonists (e.g. warfarin), heparin and low molecular            weight analogues thereof, factor Xa inhibitors, and direct            thrombin inhibitors (e.g. argatroban); antiplatelet agents,            such as cyclooxygenase inhibitors (e.g. aspirin), adenosine            diphosphate (ADP) receptor inhibitors (e.g. clopidogrel),            phosphodiesterase inhibitors (e.g. cilostazol), glycoprotein            IIB/IIA inhibitors (e.g. tirofiban), and adenosine reuptake            inhibitors (e.g. dipyridamole);        -   anti-obesity agents, such as appetite suppressant (e.g.            ephedrine), including noradrenergic agents (e.g.            phentermine) and serotonergic agents (e.g. sibutramine),            pancreatic lipase inhibitors (e.g. orlistat), microsomal            transfer protein (MTP) modulators, diacyl            glycerolacyltransferase (DGAT) inhibitors, and cannabinoid            (CB1) receptor antagonists (e.g. rimonabant);        -   feeding behavior modifying agents, such as orexin receptor            modulators and melanin-concentrating hormone (MCH)            modulators;        -   neuropeptide Y (NPY)/NPY receptor modulators;        -   pyruvate dehydrogenase kinase (PDK) modulators;        -   serotonin receptor modulators;        -   leptin/leptin receptor modulators;        -   ghrelin/ghrelin receptor modulators;    -   an agent that enhances Beta-cell function;    -   an agent that stimulates energy expenditure (e.g.        beta-adrenergic stimulants, UCP-1 agonists, brown fat modulators        and stimulants);    -   an agent that induces lysis of adipocytes (e.g. an antibody);    -   nicotine or a nicotine withdrawal aid;    -   estrogen, a natural or synthetic modulator of an estrogen        receptor;    -   a μ-opioid receptor modulator; and    -   monoamine transmission-modulating agents, such as selective        serotonin reuptake inhibitors (SSRI) (e.g. fluoxetine),        noradrenaline reuptake inhibitors (NARI),        noradrenaline-serotonin reuptake inhibitors (SNRT), triple        monoamine reuptake blockers (e.g. tesofensine), and monoamine        oxidase inhibitors (MAOI) (e.g. toloxatone and amiflamine), or a        pharmaceutically acceptable salt thereof.

In an example embodiment, an MC4R agonist (e.g., an MC4R agonist that ispart of an ionic complex or uncomplexed) and a second agent areadministered with the simultaneous, sequential or separateadministration of a very low calorie diets (VLCD) or low-calorie diets(LCD).

Method of Preparing the Ionic Complex

The invention further relates to a method of for preparing the ioniccomplex and pharmaceutical composition of the invention. The methodincludes providing a cationic polypeptide and an anionic excipientselected from a PEG-carboxylic acid, a fatty acid having 10 or morecarbon atoms, a phospholipid, or a combination thereof. Combining thecationic polypeptide and the anionc excipient under conditions to forman ionic complex and preparing a pharmaceutical composition comprisingthe ionic complex.

For example, a mixture of an anionic excipient selected from aPEG-carboxylic acid, a fatty acid having 10 or more carbon atoms, aphospholipid, or a combination thereof and any additional excipients inan aqueous medium (e.g., water) can be uniformly constituted in a welldispersed state by autoclaving the mixture under suitable conditions.Suitable conditions can include, for example, from about 3 minutes toabout 25 minutes at a temperature of 121° C. to 134° C. An example ofsuitable conditions includes, a time of about 15 minutes at atemperature of about 121° C. The use of autoclaving also affords asterile mixture. A sterile aqueous solution of the cationic peptide canthen be added to the mixture to afford, a sterile, homogeneous ioniccomplex of this invention. Depending on the nature of the anionicexcipient, either a clear solution or a uniform suspension of the ioniccomplex can be obtained. For example, the PEG-carboxylate andmPEG2000-DSPE used in the examples described herein typically results ina clear solution comprising the ionic complex, while the use of DPPAlipid typically results in a homogenous suspension comprising the ioniccomplex.

Alternatively, the mixture of the ionic complex can be prepared bydissolving the excipients in water with the cationic polypeptide andsterile filtering the resulting mixture through a 0.2 micron filter.

A method of making an ionic complex comprising a cationic polypeptideand an anionic excipient selected from: a PEG-carboxylic acid; a fattyhaving 10 or more carbon atoms; a phospholipid; and a combinationthereof, comprising:

-   -   a) preparing a mixture of the anionic excipient and an aqueous        excipient diluent;    -   b) autoclaving the mixture under conditions sufficient to        sterilize the excipient;    -   c) adding a sterile peptide solution comprising a cationic        polypeptide and a aqueous peptide diluent to the excipient        mixture.

In one embodiment, the excipient mixture is a suspension. In anotherembodiment, the excipient mixture is a solution.

In an alternative embodiment, the invention relates to a method ofmaking an ionic complex comprising a cationic polypeptide and an anionicexcipient selected from: a PEG-carboxylic acid; a fatty having 10 ormore carbon atoms; a phospholipid; and a combination thereof,comprising:

-   -   a) preparing a solution of the anionic excipient and an aqueous        excipient diluent;    -   b) filtrating the solution of step a through a 0.2 micron        filter;    -   c) adding a sterile peptide solution comprising a cationic        polypeptide of and an aqueous peptide diluent to the excipient        solution of step b.

In yet another embodiment, the invention relates to a method of makingan ionic complex comprising a cationic polypeptide and an anionicexcipient selected from: a PEG-carboxylic acid; a fatty having 10 ormore carbon atoms; a phospholipid; and a combination thereof,comprising:

-   -   a) preparing a solution comprising the anionic excipient an        aqueous excipient diluent, and a cationic polypeptide;    -   b) sterilizing the resulting solution by filtering through a 0.2        micron filter.

Suitable examples of excipient and cationic polypeptide excipientinclude polyol (e.g., propylene glycol, tripropylene glycol, glycerol,ethanol, benzyl alcohol, DMSO, NMP, DMF, water, pH stabilizing bufferedsolutions and mixtures thereof.

Exemplification Peptide Synthesis

The cationic polypeptides suitable for use in this invention wereprepared by conventional solid phase peptide synthesis. For example,Peptide 1 and other MC4R analogs described herein can be preparedaccording to the methods described in U.S. Pat. No. 8,349,797, theentire content of which is hereby incorporated by reference. The peptidechain was elongated in a step-wise manner starting with its C-terminalend amino acid derivative coupled on to an appropriately selected solidsupport resin known to be suitable for peptide synthesis. For thesynthesis of peptide with a C-terminal amide function, Rink amide MBHAresin was employed as solid support. For the synthesis of peptides withthe C-terminal free carboxyl function, resins such as 2-chlorotritylchloride resin, Wang, or Merrifield resin may be utilized that form anester bond with the Fmoc-amino acid. Most of these ester linkedFmoc-amino acid-resin types are commercially available from varioussources and generally used when feasible.

Synthesis of Disulfide-Cyclized Peptides

The linear derivative of a disulfide cyclic peptides amide was assembledusing Fmoc chemistry on a solid-phase peptide synthesizer. The Fmoc-Rinkamide resin was placed in a reaction vessel and swollen with NMP. It wasthen treated with 20% piperidine in NMP for 15 minutes, followed by 3washes of NMP. The resin was tested for positive Kaiser's test (Kaiser,E., Colescot, R. L., Bossinge, C. D. & Cook, P. I. Anal. Biochem., 1990,34: 595-598). It was resuspended in NMP and mixed with the requiredfirst C-terminal Fmoc-amino acid derivative and HOBt. The couplingreaction was started by the addition of HBTU reagent and DIPEA. Aftermixing for 2-3 hours, the completion of coupling was confirmed by anegative Kaiser's test on a small aliquot of the resin withdrawn fromthe reaction mixture. The resin was then washed three times with NMP.Thereafter, the Fmoc group was removed as described earlier and thewhole cycle repeated with the second C-terminal Fmoc-amino acidderivative as described. The same cycle of reactions was repeatedsequentially with each of the incoming amino acid. The chloranil colortest (Vojkovsky, T. Pept. Res., 1995, 8: 236-237) was used instead ofKaiser's test for positive testing of Fmoc deprotection from the prolineresidue in the peptide sequence as well for testing completion ofcoupling of an amino acid to proline (a negative chloranil test). Incase of peptides with N-terminal acetyl group, the Fmoc deprotectedpeptide resin was treated for 10 minutes with acetic anhydride andpyridine. The resin after testing negative for Kaiser's test was washedwith NMP, dichlormethane and dried in vacuo. The Fmoc-amino acidderivatives were used for the synthesis of these peptides. Thetrifunctional amino acid derivatives used were the following:Fmoc-Cys(Trt)-OH, Fmoc-Trp(Boc)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-His(Trt)-OH,Fmoc-Asn(Trt)-OH, Fmoc-Gln(Trt)-OH, Fmoc-hCys(Trt)-OH, Fmoc-Pen(Trt)-OH,Fmoc-Tyr(But)-OH, Fmoc-His(1-Me)-OH, Fmoc-His(3-Me)-OH, andFmoc-Glu(OBut)-OH.

To cleave the peptide off the resin as well as to deprotect the sidechain functions, the peptide resin was taken in: 2% TIS/5% water/5%(w/v) DTT/88% TFA. The solution was allowed to mix for 3.5 hours andthen filtered. The filtrate was mixed with cold anhydrous ethyl ether.The precipitate was collected by centrifugation. The solvent wasdecanted and the peptide pellet was re-suspended in fresh ether. Theether workup was repeated two more times. The peptide was dried invacuo. The crude linear peptide product was diluted to a concentrationof 2 mg/mL in 5% acetic acid and 0.5M iodine/methanol was added dropwisewith vigorous stirring until a persistent pale yellow color of thesolution was achieved. The solution was stirred for additional 10minutes. Excess iodine was then quenched by adding IM sodium thiosulfateunder mixing until the mixture was rendered colorless. The cyclizedpeptide solution was lyophilized and the crude powder purified bypreparative HPLC using a reversed-phase C-18 column. The purifiedproduct fractions were pooled and lyophilized. The peptides wereanalyzed by mass spectrometry using electrospray ionization techniqueand identified to correct mass.

Synthesis of Lactm-Cyclized Peptides

The cyclic lactam peptides were also synthesized by standard solid phasepeptide synthesis methods. For peptides with a C-terminus Dpr, anFmoc-Dpr(Mtt)-BHA resin was transferred to a solid phase peptidesynthesizer reactor. The Fmoc group, was removed as described above andthe next Fmoc-protected amino acid, such as for exampleFmoc-Trp(Boc)-OH, was coupled to the resin through standard couplingprocedures. The Fmoc protective group was removed and the remainingamino acids added individually in the correct sequence, by repeatingcoupling and deprotection procedures until the amino acid sequence wascompleted. For glutamic acid, coupling Fmoc-Glu(OPip) was employed. Thefully assembled peptide was then acetylated at the N-terminus as permethod described earlier for the disulfide series of peptides. Theorthogonally protected side chains were then removed. For example, apeptide resin with either an orthogonally protected side chain of Glu as2-phenylisoproply (OPip) ester or Dpr as 4-methyltrityl (Mtt), werecleaved by treatment with 1% TFA in dicholoromethane. The deprotectedpeptide resin was suspended in NMP, and treated with HBTU/DIPEA. Aftercyclization (a negative Kaiser's test), the peptide-resin was washedwith DCM and dried. The cyclic peptide was cleaved from the resin alongwith any remaining protective groups using trifluoroacetic acid (TFA) inthe presence of water and 1,2-ethanedithiol (EDT). The product wascollected by precipitation upon the addition of cold anhydrous ether andcollected by centrifugation. Final purification was by reversed phaseHPLC using a reversed phase C-18 column. The purified peptide collectedby lyophilization and analyzed for its mass by mass spectrometry usingelectron spray methodology.

TABLE 1 Examples of Cationic Peptides of Formula I Compound (SEQ IDNO: 1) Peptide 1

 1 Ac-Arg-cyclo[Cys-D-Leu-His-D-Phe-Arg-Trp-Cys]-NH₂ (SEQ ID NO: 2)  2Ac-Arg-cyclo[Cys-D-Ile-His-D-Phe-Arg-Trp-Cys]-NH₂ (SEQ ID NO: 3)  3Ac-Arg-cyclo[Cys-D-Tie-His-D-Phe-Arg-Trp-Cys]-NH₂   Tie = t-butylglycine (SEQ ID NO: 4)  4Ac-Arg-cyclo[Cys-D-Val-His-D-Phe-Arg-Trp-Cys]-NH₂ (SEQ ID NO: 5)  5Ac-Arg-cyclo[Cys-D-Ala-His(3-Me)-D-Phe-Arg-Trp-Cys]-NH₂ (SEQ ID NO: 6) 6 Ac-Arg-cyclo[Cys-D-Ala-His(1-Me)-D-Phe-Arg-Trp-Cys]-NH₂ (SEQ ID NO:7)  7 Ac-Arg-cyclo[Cys-D-Ala-Trp-D-Phe-Arg-Trp-Cys]-NH₂ (SEQ ID NO: 8) 8 Ac-Arg-cyclo[Cys-D-Ala-Gln-D-Phe-Arg-Trp-Cys]-NH₂ (SEQ ID NO: 9)  9Ac-Arg-cyclo[Cys-D-Ala-Asn-D-Phe-Arg-Trp-Cys]-NH₂ (SEQ ID NO: 10) 10Ac-Arg-cyclo[Cys-D-Ala-Arg-D-Phe-Arg-Trp-Cys]-NH₂ (SEQ ID NO: 11) 11Ac-Arg-cyclo[Cys-D-Ala-Tyr-D-Phe-Arg-Trp-Cys]-NH₂ (SEQ ID NO: 12) 12Ac-Arg-cyclo[Cys-D-Ala-D-Pro-D-Phe-Arg-Trp-Cys]-NH₂ (SEQ ID NO: 13) 13Ac-Arg-cyclo[Cys-D-Ala-Pro-D-Phe-Arg-Trp-Cys]-NH₂ (SEQ ID NO: 14) 14Ac-Arg-cyclo[Cys-D-Ala-Pro-D-Phe(p-F)-Arg-Trp-Cys]-NH₂ (SEQ ID NO: 15)S1 Ac-Arg-cyclo[Cys-D-Ala-Atc-D-Phe-Arg-Trp-Cys]-NH₂ (SEQ ID NO: 16) Atc=

S2 Ac-Arg-cyclo[Cys-D-Ala-QAla-D-Phe-Arg-Trp-Cys]-NH₂ (SEQ ID NO: 17)QAla =

S3 Ac-Arg-cyclo[Cys-D-Ala-sChp-D-Phe-Arg-Trp-Cys]-NH2 (SEQ ID NO: 18)sChp =

S4 Ac-Arg-cyclo[Cys-D-Ala-X-D-Phe-Arg-Trp-Cys]-NH₂ (SEQ ID NO: 19) X =

15 Ac-Arg-cyclo[hCys-Ala-D-Phe-Arg-Trp-Cys]-NH₂ (SEQ ID NO: 20) 16Ac-Arg-cyclo[hCys-D-Ala-D-Phe-Arg-Trp-Cys]-NH₂ (SEQ ID NO: 21) 17Ac-Arg-cyclo[hCys-D-Ala-D-Phe-Arg-Trp-Pen]-NH₂ (SEQ ID NO: 22) 18Ac-Arg-cyclo[Glu-D-Ala-D-Phe-Arg-Trp-Dpr]-NH₂ (SEQ ID NO: 23) 19Ac-Arg-cyclo[Glu-Ala-D-Phe-Arg-Trp-Dpr]-NH₂ (SEQ ID NO: 24) S5Ac-Arg-cyclo[hCys-Aib-D-Phe-Arg-Trp-Cys]-NH₂ (SEQ ID NO: 25) S6Ac-Arg-cyclo[hCys-Sar-D-Phe-Arg-Trp-Cys]-NH₂ (SEQ ID NO: 26) S7Ac-Arg-cyclo[hCys-Val-D-Phe-Arg-Trp-Cys]-NH₂ (SEQ ID NO: 27) S8Ac-Arg-cyclo[hCys-D-Val-D-Phe-Arg-Trp-Cys]-NH₂ (SEQ ID NO: 28) S9Ac-Arg-cyclo[hCys-Gln-D-Phe-Arg-Trp-Cys]-NH₂ (SEQ ID NO: 29) S10Ac-Arg-cyclo[hCys-D-Gln-D-Phe-Arg-Trp-Cys]-NH₂ (SEQ ID NO: 30) S11Ac-Arg-cyclo[hCys-Ala-D-Phe-Arg-Trp-Pen]-NH₂ (SEQ ID NO: 31) S12Ac-Arg-cyclo[D-Pen-D-Ala-D-Phe-Arg-Trp-hCys]-NH₂ (SEQ ID NO: 30) S13Ac-Arg-cyclo[Cys-D-Ala-D-Phe-Arg-Trp-hCys]-NH₂ (SEQ ID NO: 33) S14Ac-Arg-cyclo[Pen-D-Ala-D-Phe-Arg-Trp-hCys]-NH₂ (SEQ ID NO: 34) S15Ac-Arg-cyclo[D-hCys-D-Ala-D-Phe-Arg-Trp-Cys]-NH₂ (SEQ ID NO: 35) D1Ac-Arg-cyclo[hCys-Pro-D-Phe-Arg-Trp-Cys]-NH₂ (SEQ ID NO: 36) D2Ac-Arg-cyclo[hCys-D-Pro-D-Phe-Arg-Trp-Cys]-NH₂ (SEQ ID NO: 37) 20Ac-Arg-cyclo[Cys-Val-Gln-D-Phe-Arg-Trp-Cys]-NH₂ (SEQ ID NO: 38) 21Ac-Arg-cyclo[Cys-D-Val-Gln-D-Phe-Arg-Trp-Cys]-NH₂ (SEQ ID NO: 39) D3Ac-Arg-cyclo[Cys-D-Val-His(1-Me)-D-Phe-Arg-Trp-Cys]-NH₂ (SEQ ID NO: 40)D4 Ac-TzAla-cyclo[Cys-Ala-Gln-D-Phe-Arg-Trp-Cys]-NH2 (SEQ ID NO: 41)TzAla = 3-(1,2,4-triazol-1-yl)-L- Ala

22 Ac-Glu-cyclo[Cys-Ala-His-D-Phe-Arg-Trp-Cys]-NH₂ (SEQ ID NO: 42) 23(SEQ ID NO: 43)

24 (SEQ ID NO: 44)

25 (SEQ ID NO: 45)

Certain cationic polypeptides of Table 1 were tested to assess activityin the assays described below. The data is provided in Table 2.

Peptide Testing: Radioligand Binding Assays:

Receptor binding assays for determining the binding constant (K_(d)) orinhibition concentration (IC₅₀) for displacing a radio-labeled ligandfrom the receptor of a cyclic peptide of the invention may be conductedby any means known in the art.

As an example, the cell membrane preparations for a binding assay areprepared from CHO-K1 cells transfected to stably express hMC receptorsubtypes 1, 3, 4 or 5. Competitive inhibition of[¹²⁵I](Tyr²)-(Nle⁴-D-Phe⁷)-alpha-MSH ([¹²⁵I]-NDP-α-MSH binding iscarried out in polypropylene 96 well plates. Briefly, the cell membranes(1-10 μg protein/well), prepared as described above, is incubated in 50mM Tris-HCl at pH 7.4 containing 0.2% BSA, 5 mM MgCl₂, 1 mM CaCl₂) and0.1 mg/mL bacitracin, with increasing concentrations of the testcompound and 0.1-0.3 nM [¹²⁵I]-NDP-α-MSH for approximately 120 minutesat 37° C. Bound [¹²⁵I]-NDP-α-MSH ligand is separated from free[¹²⁵I]-NDP-α-MSH by filtration through GF/C glass fiber filter plates(Unifilter®, Meriden, Conn., USA) presoaked with 0.1% (w/v)polyethylenimine (PEI). Filters are washed three times with 50 mMTris-HCl at pH 7.4 at a temperature of approximately 0-4° C. and thenassayed for radioactivity. The binding data are analyzed bycomputer-assisted non-linear regression analysis.

Cyclic AMP Stimulation Assay:

Functional assays to determine agonist or antagonist status of a cyclicpeptide of the invention may be conducted by any means known in the art.

Electrochemiluminescence (ECL) Assay

Stimulation of intracellular cyclic AMP (cAMP) levels by the peptides isdetermined in a dose dependent manner by an electrochemiluminescence(ECL) assay (Meso Scale Discovery, Gaithersburg, Md., USA; referred tohereinafter as “MSD”). Briefly, the CHO-K1 cells stably expressing thehMC receptor subtypes are suspended in RMPI 1640® assay buffer (RMPI1640 buffer contains 0.5 mM IBMX, and 0.2% protein cocktail (MSD blockerA)). About 7,000 cells/well of the transgenic CHO-K1 cells stablyexpressing hMC receptor subtypes 1, 3, 4 or 5 are dispensed in 384-wellMulti-Array plates (MSD) containing integrated carbon electrodes andcoated with anti-cAMP antibody. Increasing concentrations of the testcompounds are added and the cells are incubated for approximately 40minutes at 37° C. A cell lysis buffer (HEPES-buffered saline solutionwith MgCl₂ and Triton X-100® at pH 7.3) containing 0.2% protein cocktailand 2.5 nM TAG™ ruthenium-labeled cAMP (MSD) is added and the cells areincubated for approximately 90 minutes at room temperature. At the endof the second incubation period, the read buffer (Tris-buffered solutioncontaining an ECL co-reactant and Triton X-100 at pH 7.8) is added andthe cAMP levels in the cell lysates are immediately determined by ECLdetection with a Sector Imager 6000 Reader® (MSD). Data are analyzedusing a computer-assisted non-linear regression analysis (XL fit; IDBS)and reported as either an EC₅₀ value. The EC₅₀ represents theconcentration of an agonist compound needed to obtain 50% of the maximumreaction response, e.g., 50% of the maximum level of cAMP as determinedusing the assay described above.

cAMP Measurement Assay:

Human MC4-R transfected cells are grown to confluence in 96 well plates(plating approximately 250,000 cells per well). The cells are treated intriplicate sets with 0.2 mM isobutylmethylxanthine (IBMX) and gradedconcentrations of the peptide or alternatively the peptide in thepresence of 20 nM NDP-MSH. Cells similarly treated but with only 20 nMNDP-MSH serve as positive controls in a volume of 200 μL. A buffer blankserving as a negative control is also included. After incubation of onehour at 37° C., the cells are lysed by the addition of 50 μL of a celllysis buffer. Total cAMP accumulated in 250 μL of this incubation mediumis quantitated using a commercially available low pH cAMP assay kit(Amersham BioSciences) as per procedure specified by the kit supplier. Apeptide showing cAMP accumulation in the range same or higher than thealpha-MSH as positive control is considered to be an agonist. The datafor agonist is plotted and curve fitted to determine the EC₅₀ value. Apeptide showing accumulation in the same range as the negative control(buffer blank in the absence of alpha-MSH) is ineffective at the testconcentration. A peptide showing attenuated accumulation is consideredto be an antagonist if there is inhibition in cAMP when alpha-MSH isalso present in the assay. Similar assay can be performed with hMC-1R,hMC-3R, and hMC-5R cells.

cAMP Accumulation Measurement via a β-galactosidase (β-Gal) ReporterSystem:

A chemiluminescence readout system that uses an enzyme fragmentcomplementation (EFC) system with β-galactosidase (β-Gal) as thefunctional reporter system was used. This assay system for variousmelanocortin receptor systems is commercially available (cAMP HunterGPCR assay system, Discoverx Corp, Fremont, Calif.). This assay utilizesthe β-Gal enzyme that is split into two complementary portions; EA forEnzyme Acceptor and ED for Enzyme Donor. In the assay, the ED portionfused to cAMP is made to compete with cAMP generated by cells forbinding to a cAMP-specific antibody. The EA is then added to form activeβ-Gal with any unbound ED-cAMP. This active enzyme then converts achemiluminescent substrate to generate an output signal that is recordedon a standard microplate reader.

Briefly, 10000 cells per well are plated overnight and each well (cellsincubated with 10 μl assay buffer) is then incubated with 4× serialconcentration of the test compound in the cell assay buffer (5 μL) andcAMP antibody reagent (5 μL) for 30 min at 37° C. The cell lysis buffer(20 μL) containing ED-cAMP coupled enzyme fragment and the reportersubstrate (Emerald II-Galacton Star, 5:1) is then added and incubatedfor 60 min at room temperature. Next, 20 μL of EA β-Gal fragment reagentis added. After further incubation for 120 min at room temperature, thechemiluminescence is measured by a plate reader (Envision), and the dataused to calculate EC₅₀ values for the test peptide.

The results are presented in Table 2.

TABLE 2 EC50 (nM) values of example cationic polypeptides useful in theinvention cAMP Assay (EC-50) Ratios of EC50 values Compound MC1R MC3RMC4R MC5R MC1/4 MC3/4 MC5/4 PEPTIDE 1 5.8 5.3 0.3 1600 20 20 5333 1 0.470.79 0.70 91 0.68 1.13 130 2 0.69 0.96 1 420 0.69 0.96 420 3 1 0.7 0.7672 1 1 930 4 1.25 1.59 1.34 782 0.93 1.19 584 5 4.1 405.8 1.15 1085 4350 945 6 30.4 4.3 0.7 467 40 6 662 7 273 >10 uM 34 259 8 >290 7 8 718.6 1.6 255 40 5 155 9 248 81 3 1490 90 30 530 10 6.2 3.9 2.7 2.31 1.4511 300.9 >1000 45.1 6.67 >22.2 12 13 280 >10 uM 56 707 5 >200 13 14169 >10 uM 24 283 7 >400 12 15 4 1 0.26 42 15 3.8 161 16 888 31587.5 >10000 120 420 >1338 17 195 233 13.7 2181 15 17 159 22 1.7 9.9 <0.51282 >3 >20 >2563

Preparation of the Ionic Complex and Pharmaceutical Composition of theInvention Example 1: Preparation of Formulation 2A (Peptide 1 andmPEG-10,000-Monocarboxylate)

A mixture of mPEG-10,000-mono-carboxylate (5.4 g) in 8.4 g of water wastaken in a vial. The vial was stoppered, sealed and autoclaved for 15min at 121° C. to give a uniform clear viscous solution. Upon coolingthe vial was transferred to an aseptic flow hood. The vial was opened,and a sterile aqueous solution of Peptide 1 (100 mg in 1 mL), that waspre-filtered through a 0.2 μm filter, was mixed to it. The formulatedpeptide was obtained as a uniform clear viscous solution. The HPLCanalysis assayed the Peptide 1 concentration of 9.9 mg/mL.

Example 2: Preparation of Formulation 2B (Peptide 1 andPEG-10,000-Dicarboxylate)

A mixture of PEG-10,000-dicarboxylate (2.7 g) in 2.0 g of water wastaken in a vial. The vial was stoppered, sealed and autoclaved for 15min at 121° C. to give a uniform clear viscous solution. Upon coolingthe vial was transferred to an aseptic flow hood. The vial was opened,and a sterile aqueous solution of Peptide 1 (50 mg in 0.5 mL), that waspre-filtered through a 0.2 μm filter, was mixed to it. The formulatedpeptide was obtained as a uniform clear viscous solution. The HPLCanalysis assayed the Peptide 1 concentration of 10.8 mg/mL.

Example 3: Preparation of Formulation 2C (Peptide 1 andmPEG-20,000-Mono-Carboxylate)

A mixture of PEG-m20,000-carboxylate (1.8 g) in 3.5 g of propyleneglycol-ethanol-water mixture (40:15:45 ratio v/v) was taken in a vial.The vial was stoppered, sealed and autoclaved for 15 min at 121° C. togive a uniform clear viscous solution. Upon cooling the vial wastransferred to an aseptic flow hood. The vial was opened, and a sterileaqueous solution of Peptide 1 (30 mg in 0.5 mL), that was pre-filteredthrough a 0.2 μm filter, was mixed to it. The formulated peptide wasobtained as a uniform clear viscous solution. The HPLC analysis assayedthe Peptide 1 concentration of 6 mg/mL.

Example 4: Preparation of Formulation 2D (Peptide 1 and Sodium DPPA)

A mixture of sodium DPPA (302 mg) in 8.6 g of 1:1 propylene glycol-watermixture was taken in a vial. The vial was stoppered, sealed andautoclaved for 15 min at 121° C. to yield a uniform lipid dispersion.Upon cooling the vial was transferred to an aseptic flow hood. The vialwas opened, and a sterile aqueous solution of Peptide 1 (100 mg in 1mL), that was pre-filtered through a 0.2 μm filter, was mixed to it. Theformulated peptide was obtained as a uniform non-viscous dispersion. TheHPLC analysis assayed the Peptide 1 concentration in the formulation of9.6 mg/mL.

Example 5: Preparation of Formulation 2E (Peptide 1 and Sodium Stearate)

A mixture of mannitol (300 mg) and sodium stearate (165 mg) was taken in8.3 g of water in a vial. The vial was stoppered and heated at 75° C.for 0.5 hour to give a clear solution. Upon cooling about 400 μL of 1.0Nacetic acid was added to adjust pH to be within the 6-7 range. The vialwas stoppered, sealed and autoclaved for 15 min at 121° C. Upon coolingthe vial was transferred to an aseptic flow hood. The vial was opened,and a sterile aqueous solution of Peptide 1 (100 mg in 1.0 g aqueoussolution), that was pre-filtered through a 0.2 μm filter, was mixed toit. The formulated peptide was obtained as a uniform light milky whiteopaque non-viscous suspension. The HPLC analysis assayed the Peptide 1concentration of 9.7 mg/mL.

Example 6: Preparation of Formulation #3B (Peptide 1 with SodiumStearate and mPEG-2,000-DSPE)

A mixture of mPEG-2000-DSPE (1.85 g) and sodium stearate (100.1 mg)taken in 10.8 mL of water in a vial. The vial was stoppered and heatedat 75° C. for 1 hour. Upon cooling about 240 μL of acetic acid was addedto adjust pH to be within the 6-7 range. The vial was stoppered, sealedand autoclaved for 15 min at 121° C. Upon cooling the vial wastransferred to an aseptic flow hood. The vial was opened, and a sterileaqueous solution of Peptide 1 (120 mg in 1.2 g), that was pre-filteredthrough a 0.2 μm filter, was mixed to it. The formulated peptide wasobtained as a uniform light milky white opaque non-viscous suspension.The HPLC analysis assayed the Peptide 1 concentration of 10.1 mg/mL.

Example 7: Preparation of Formulation #3C (Peptide 1 and Sodium DPPA andPEG-10,000-Dicarboxylate)

A mixture of PEG-10,000-dicarboxylate (1.68 g) and sodium DPPA (362.4mg) taken in 9.1 mL of water in a vial. The vial was stoppered, sealedand autoclaved for 15 min at 121° C. to give a uniform milky whiteopaque suspension. Upon cooling the vial was transferred to an asepticflow hood. The vial was opened, and a sterile aqueous solution ofPeptide 1 (120 mg in 1.2 g), that was pre-filtered through a 0.2 μmfilter, was mixed to it. The formulated peptide was obtained as auniform milky white opaque suspension. The HPLC analysis assayed thePeptide 1 concentration of 10.6 mg/mL.

Example 8: Preparation of Formulation #3D (Peptide 1 and Sodium DPPA andPEG-3,350)

A mixture of PEG-3,350 (1.8 g contained in 10.8 mL aqueous solution) andsodium DPPA (363 mg) was taken in a vial. The vial was stoppered, sealedand autoclaved for 15 min at 121° C. to give a uniform milky whiteopaque suspension. Upon cooling the vial was transferred to an asepticflow hood. The vial was opened, and a sterile aqueous solution ofPeptide 1 (120 mg in 1.2 g), that was pre-filtered through a 0.2 μmfilter, was mixed to it. The formulated peptide was obtained as auniform milky white opaque suspension. The HPLC analysis assayed thePeptide 1 concentration of 10.1 mg/mL.

Example 9: Preparation of Formulation #3E (Peptide Land Sodium DPPA,PEG-3,350, and Sodium CMC)

An aqueous solution of sodium CMC (Av 1\4W 90,000), (72 mg in 9.0 mL)was mixed with PEG-3,350 (1.8 g). To the resulting clear solution sodiumDPPA (216 mg) was added. The vial was stoppered, sealed and autoclavedfor 15 min at 121° C. to give a uniform milky white opaque suspension.Upon cooling the vial was transferred to an aseptic flow hood. The vialwas opened, and a sterile aqueous solution of Peptide 1 (120 mg in 1.2g), that was pre-filtered through a 0.2 μm filter, was mixed to it. Theformulated peptide was obtained as a uniform milky white opaquesuspension. The HPLC analysis assayed the Peptide 1 concentration of10.9 mg/mL.

Example 10: Preparation of Formulation #3F (Peptide 1 andmPEG-2,000-DSPE and Sodium CMC)

An aqueous solution of sodium CMC (Av 1\4W 90,000), (72 mg in 10.8 mL or6.7 mg/mL) was mixed with mPEG-2,000-DSPE (1.85 g). The vial wasstoppered, sealed and autoclaved for 15 min at 121° C. to give a uniformclear solution. Upon cooling the vial was transferred to an aseptic flowhood. Alternatively, the contents of the vial containing the mixture ofsodium CMC and mPEG-2,000-DSPE were mixed overnight to give rise to aclear solution that was sterile filtered through a 0.2 μm filter. Thevial was opened inside an aseptic flow hood, and a sterile aqueoussolution of Peptide 1 (120 mg in 1.2 g), that was pre-filtered through a0.2 μm filter, was mixed to it. The formulated peptide was obtained as auniform clear solution. The HPLC analysis assayed the Peptide 1concentration of 10.5 mg/mL.

Example 11: Preparation of Formulation #4B (Peptide 1 andmPEG-2,000-DSPE)

A mixture of mPEG-2,000-DSPE (1.855 g) and mannitol (181 mg) was takenin 8.8 mL of water for injection (nitrogen purged for 10 min) in a vial.The vial was stoppered and gently rotated to completely wet all thesolid ingredients with water. It was then autoclaved for 15 min at 121°C. The contents of the vial at this stage were a clear solution. Uponcooling the vial was transferred to an aseptic flow hood. The vial wasopened, and a sterile aqueous solution of Peptide 1 (120 mg in 1.2 g),that was pre-filtered through a 0.2 μm filter, was mixed to it for 1 h.The formulated peptide was obtained as a uniform clear solution. TheHPLC analysis assayed the Peptide 1 concentration of 8.7 mg/mL.

Example 12: Preparation of Formulation #4C (Peptide 1 andmPEG-2,000-DSPE and CMC)

An aqueous solution of sodium CMC (Av MW 90,000), at a concentration of8.9 mg/mL was prepared in nitrogen purged water for injection. To 9.8 mLof this solution taken in a vial was added mPEG-2,000-DSPE (1.853 g) andmannitol (182 mg). The vial was stoppered and gently rotated tocompletely wet all the solid ingredients with the solvent. It was thenautoclaved for 15 min at 121° C. The contents of the vial at this stagewere a clear solution. Upon cooling the vial was transferred to anaseptic flow hood. The vial was opened, and a sterile aqueous solutionof Peptide 1 (120 mg in 1.2 g), that was pre-filtered through a 0.2 μmfilter, was mixed to it for 1 h. The formulated peptide was obtained asa uniform clear solution. The HPLC analysis assayed the Peptide 1concentration of 8.0 mg/mL.

Example 13: Preparation of Formulation #4D (Peptide 1 andmPEG-2,000-DSPE and DPPA)

A mixture of mPEG-2,000-DSPE (1.86 g), sodium DPPA (363 mg), andmannitol (60.2 mg) was taken in 9 mL of water for injection (nitrogenpurged for 10 min) in a vial. The vial was stoppered and gently rotatedto completely wet all the solid ingredients with water. It was thenautoclaved for 15 min at 121° C. The contents of the vial at this stagewere a light milky white homogenous suspension. Upon cooling the vialwas transferred to an aseptic flow hood. The vial was opened, and asterile aqueous solution of Peptide 1 (120 mg in 1.2 g), that waspre-filtered through a 0.2 μm filter, was mixed to it for 1 h to give auniform light milky white opaque non-viscous suspension. The HPLCanalysis assayed the Peptide 1 concentration of 9.2 mg/mL.

Example 14: Preparation of Formulation #4E (Peptide 1 andmPEG-2,000-DSPE and CMC)

An aqueous solution of sodium CMC at a concentration of 7.2 mg/mL wasprepared in nitrogen purged water for injection. To 10 mL of thissolution taken in a vial was added mPEG-2,000-DSPE (0.94 g) and mannitol(181.7 mg). The vial was stoppered and gently rotated to completely wetall the solid ingredients with the solvent. It was then autoclaved for15 min at 121° C. The contents of the vial at this stage were a clear,colorless solution. Upon cooling the vial was transferred to an asepticflow hood. The vial was opened, and a sterile aqueous solution ofPeptide 1 (120 mg in 1.2 g), that was pre-filtered through a 0.2 μmfilter, was mixed to it for 1 h. The formulated peptide was obtained asa uniform clear solution. The HPLC analysis assayed the Peptide 1concentration of 9.6 mg/mL.

Example 15: Preparation of Formulation #4F (Peptide 1 andmPEG-2,000-DSPE and DPPA)

A mixture of mPEG-2,000-DSPE (0.954 g), sodium DPPA (183.7 mg), andmannitol (61.7 mg) was taken in 9.6 mL of water for injection (nitrogenpurged for 10 min) in a vial. The vial was stoppered and gently rotatedto completely wet all the solid ingredients with water. It was thenautoclaved for 15 min at 121° C. The contents of the vial at this stagewere a light milky white homogenous suspension. Upon cooling the vialwas transferred to an aseptic flow hood. The vial was opened, and asterile aqueous solution of Peptide 1 (120 mg in 1.2 g), that waspre-filtered through a 0.2 μm filter, was mixed to it for 1 h to obtaina uniform light milky white opaque non-viscous suspension. The HPLCanalysis assayed the Peptide 1 concentration of 9.9 mg/mL.

Example 16: Preparation of Formulation Having Peptide 1,mPEG-2,000-DSPE, CMA and D-Mannitol

Strength Peptide 1 10 mg/mL* mPEG2000-DSPE · Na 100 mg/mL CMC · Na (AvMW, 90K) 8 mg/mL D-Mannitol 22 mg/mL Water As needed to 1 mL finalvolume *Based on peptide content

The following preparation is based on a 10 mL formulation and can beeasily scaled down to provide the 1 mL final volume in the table above.

An aqueous solution of sodium CMC (Av 1\4W 90,000), (80 mg) andD-mannitol (220 mg) is taken in 7 ml of water for injection in apre-weighed vial with a stir bar and stopper. To this solution solidsodium mPEG-2,000-DSPE (1 g) is added. The vial is stoppered, andsealed. It is placed in a water bath set at 60° C. and the contentsstirred. Alternatively, the vial can be placed in an oven maintained at60° C. Initially the contents of the vial may be cloudy, but will turninto a clear uniform solution within 1-2 hours. The vial is allowed toattain room temperature. It is then opened and an aqueous solution ofPeptide 1 (100 mg in 1 mL, based on its net peptide content) is mixedinto it. The contents may turn cloudy momentarily, but will turn into aclear homogenous solution upon mixing. The vial is weighed and theweight of the total contents adjusted to 10.1 gm by adding the requiredamount of water for injection solution. The resulting formulation isthen mixed and filtered through a 0.2 μm filter inside an aseptic flowhood. The formulated peptide was obtained as a uniform clear solutionand is assayed by HPLC to confirm the 10 mg/ml concentration ofPeptide-1.

Example 17: Preparation of Formulation Having Peptide 1, Sodium DPPA,and PEG-3,350

Strength Peptide 1 10 mg/mL* DPPA · Na 30.2 mg/mL PEG-3350 120 mg/mLWater As need to 1 mL final volume *Based on peptide contentThe following preparation is based on a 10 mL formulation and can beeasily scaled down to provide the 1 mL final volume in the table above.

In a pre-weighed stoppered vial is added 1.2 gm of PEG-3350 and 8 mL ofwater for injection. The contents are dissolved by mixing and 302 mg ofsodium DPPA is added. The vial is stoppered, sealed and placed in anautoclave autoclaved for 15 min at 121° C. to give a uniform milky whiteopaque suspension. Upon cooling the vial is transferred to an asepticflow hood. The vial is opened, and a sterile aqueous solution of Peptide1 (100 mg in 1 mL water, based on its net peptide content), that waspre-filtered through a 0.2 μm filter, is mixed to it. The total contentof vial is adjusted to 10.1 gm by the addition of appropriate amount ofwater for injection. Upon mixing, the formulated peptide is obtained asa uniform milky white opaque suspension. The formulated peptide isassayed by HPLC to confirm the 10 mg/ml concentration of Peptide-1.

Example 18: Administration of Formulations to Cynomolgus Monkeys toEvaluate Pharmacokinetics

A group of Cynomolgus monkeys (average body weight range of 3 to 7 kg)were randomized into a group of 6 for each formulation described aboveand for Formulation 3A. Each monkey in the cohort was subcutaneouslyadministered in the scapular area with a single bolus dose of aformulation calculated at 0.5 mg/kg body weight. Blood samples weretaken at various time points staring at 30 minutes post dosing to 36 or48 hours. The plasma was harvested and peptide drug concentration assaysusing LC-MS/MS technique. The pharmacokinetic data is presented in Table3 below, and the pharmacokinetic profile is set forth graphically inFIG. 1, FIG. 2, and FIG. 3.

TABLE 3 Pharmacokinetic Data for Formulations (N = 6; Dose = 0.5 mg/KgControl formulations contain Peptide 1 in citrate buffer) Cmax Tmax AUClast T-1/2 Formulation Description (ng/mL) (hr) (hr * ng/mL) (hr) % FrCONTROL; SC formulation-Citrate: SC Control-D27455, (Citrate) 1080 ± 2100.50 ± 3680 ± 850 3.55 ± 0.536 100 4A 0 10 mg/ml SC Control-D27455,(Citrate) 1100 ± 101 0.50 ± 3970 ± 777 3.50 ± 0.48  100 3A 0 10 mg/mlGroup: 2 PEPTIDE 1 AND mPEG-COOH- 438 ± 62 2.17 ± 3140 ± 295 3.64 ±0.134 85.3 * 10K 0.98 2A PEPTIDE 1 AND PEG-(COOH)2-  472 ± 125 2.33 ± 3830 ± 1090 3.85 ± 1.27  104 * 10K 0.82 2B PEPTIDE 1 AND mPEG-COOH- 443 ± 116 3.33 ± 4240 ± 902 4.13 ± 0.536 115 * 20K 1.03 2C PEPTIDE 1AND DPPA/PG (PG is   351 ± 30.4  1.0 ± 3530 ± 696 6.81 ± 1.17  96 *propylene glycol as solvent) 0.55 2D PEPTIDE 1 AND Na · stearate 838 ±73  0.5 ± 2800 ± 281 3.46 ± 0.798 76 * 2E 0.0 Group: 3 PEPTIDE 1,Na·Stearate, AND   303 ± 26.4 7.33 ± 3640 ± 608 — 91.7 mPEG-DSPE 1.63 3BPEG-(COOH)2-10K/Na · DPPA   272 ± 52.4 2.00 ± 3330 ± 707 4.47 ± 1.07 83.9 AND PEPTEDE 1 0 3C PEG3350/Na · DPPA AND 228 ± 50 4.33 ± 3180 ± 5421.07 ± 0.15  80.1 PEPTEDE 1 1.97 3D PEG3350, CMC, sodium DPPA   317 ±68.4 2.50 ± 2890 ± 266 4.49 ± 0.80  72.8 AND PEPTIDE 1 1.22 3ECMC/mPEG-DSPE AND   258 ± 77.8 10.0 ± 3990 ± 788 4.53 ± 0     100.5PEPTIDE 1 2.19 3F Group: 4 mPEG-DSPE AND PEPTIDE 1,   270 ± 53.5 8.67 ±3570 ± 550 3.36 ± 0.654 96.7 4B 3.01 mPEG-DSPE/CMC AND   320 ± 49.9 11.3± 4340 ± 353 3.79 ± 0.503 117 PEPTIDE 1, 3.01 4C mPEG-DSPE/DPPA AND  283 ± 72.4 9.33 ± 3570 ± 789 3.09 ± 0.304 96.6 PEPTIDE 1, 2.07 4DmPEG-DSPE/CMC AND   292 ± 38.0 8.00 ± 3710 ± 531 3.05 ± 0.304 100PEPTIDE 1, 3.03 4E mPEG-DSPE/DPPA AND   302 ± 24.2 8.00 ± 3940 ± 2423.25 ± 0.352 106.5 PEPTEDE 1 2.53 4F * % Fr represents thebioavailability of a formulation when compared to the Control 4A.

As is evident from the results of comparative pharmacokinetic studieswith the exemplified ionic-complexes of Peptide-1, there was significantreduction in the C-max (Peptide 1 Cmax of 1100 ng/mL vs 228 ng/mLobserved with complex 3D). Many other ionic complexes of this inventiondisplayed similar range of blunted C-max as compared to Peptide-1. Inaddition to this, the T-max was also significantly increase with severalof these ionic complexes (T-max of Peptide-1 was 0.5 hrs vs. T-max ofseveral hours with many of the ionic complexes).

The teachings of all patents, published applications and referencescited herein are incorporated by reference in their entirety.

While this invention has been particularly shown and described withreferences to example embodiments thereof, it will be understood bythose skilled in the art that various changes in form and details may bemade therein without departing from the scope of the inventionencompassed by the appended claims.

1-56. (canceled)
 57. An ionic complex comprising: (i) a cationicpolypeptide represented by the following structural formula:

or a pharmaceutically acceptable salt thereof; and (ii) a1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE) conjugatedpolyethylene glycol, wherein the molar ratio of the cationic polypeptideto the 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE) conjugatedpolyethylene glycol ranges from about 1:1 to about 1:10.
 58. The ioniccomplex of claim 57, wherein the molar ratio of the cationic peptide tothe 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE) conjugatedpolyethylene glycol ranges from about 1:2 to about 1:8.
 59. The ioniccomplex of claim 57, wherein the molar ratio of the cationic peptide tothe 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE) conjugatedpolyethylene glycol ranges from about 1:3 to about 1:6.
 60. The ioniccomplex of claim 57, further comprising an anionic excipient selectedfrom carboxymethylcellulose (CMC), stearic acid, and DPPA.
 61. The ioniccomplex of claim 60, wherein the anionic excipient is CMC.
 62. The ioniccomplex of claim 60, wherein the anionic excipient is stearic acid. 63.The ionic complex of claim 60, wherein the anionic excipient is DPPA.64. The ionic complex of claim 57, wherein the1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE) conjugatedpolyethylene glycol is selected from mPEG-2,000-DSPE andmPEG-5,000-DSPE.
 65. The ionic complex of claim 64, wherein the1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE) conjugatedpolyethylene glycol is mPEG-2,000-DSPE.
 66. The ionic complex of claim57, further comprising a combination of anionic excipients.
 67. Theionic complex of claim 66, wherein the combination of anionic excipientsis selected from: stearic acid and mPEG-2,000-DSPE; DPPA andmPEG-2,000-DSPE; mPEG-2,000-DSPE and CMC; and mPEG-2,000-DSPE.
 68. Theionic complex of claim 57, wherein the ionic complex has the propertythat it forms a drug depot in a physiological environment.
 69. The ioniccomplex of claim 57, wherein the ionic complex has the property that itforms a drug depot when delivered subcutaneously.
 70. A pharmaceuticalcomposition comprising: (a) an ionic complex comprising: (i) a cationicpolypeptide represented by the following structural formula:

or a pharmaceutically acceptable salt thereof; and (ii) a1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE) conjugatedpolyethylene glycol, wherein the molar ratio of the cationic polypeptideto the 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE) conjugatedpolyethylene glycol ranges from about 1:1 to about 1:10; and (b) a thepharmaceutically acceptable carrier.
 71. The pharmaceutical compositionof claim 70, wherein the molar ratio of the cationic peptide to the1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE) conjugatedpolyethylene glycol ranges from about 1:2 to about 1:8.
 72. Thepharmaceutical composition of claim 70, wherein the ionic complexfurther comprises an anionic excipient selected fromcarboxymethylcellulose (CMC), stearic acid, and DPPA.
 73. Thepharmaceutical composition of claim 70, wherein the1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE) conjugatedpolyethylene glycol is selected from mPEG-2,000-DSPE andmPEG-5,000-DSPE.
 74. The pharmaceutical composition of claim 73, whereinthe 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE) conjugatedpolyethylene glycol is mPEG-2,000-DSPE.
 75. The pharmaceuticalcomposition of claim 70, wherein the pharmaceutically acceptable carrieris selected from a polyethylene glycol (PEG), polyol, ethanol,dimethyksulfoxide (DMSO), N-methylpyrrolidone (NMP), dimethylformamide(DMF), benzyl alcohol, water, pH stabilizing buffered solutions andmixtures thereof.
 76. The pharmaceutical composition of claim 75,wherein the pharmaceutically acceptable carrier comprises a PEG havingan average molecular weight of about 100 to about 5,000.
 77. Thepharmaceutical composition of claim 75, wherein the polyol is selectedfrom propylene glycol, tripropylene glycol, glycerol and mixturesthereof.
 78. The pharmaceutical composition of claim 70, wherein theconcentration of cationic polypeptide is from about 0.01 mg/mL to about100 mg/mL.
 79. The pharmaceutical composition of claim 70, wherein theconcentration of cationic polypeptide is from about 1 mg/mL to about 50mg/mL.
 80. The pharmaceutical composition of claim 70, comprisingmPEG-2,000-DSPE at a concentration of from about 50 mg/mL to about 200mg/mL.
 81. The pharmaceutical composition of claim 70, furthercomprising carboxymethylcellulose (CMC) at a concentration of 8 mg/mL.82. The pharmaceutical composition of claim 70, further comprisingD-mannitol.
 83. The pharmaceutical composition of claim 82, wherein theconcentration of the D-mannitol is from about 10 mg/mL to about 25mg/mL.
 84. The pharmaceutical composition of claim 70, furthercomprising a preservative.
 85. The pharmaceutical composition of claim84, wherein the preservative is selected from phenol and benzyl alcohol.86. A method of treating a subject having a disease or disorderresponsive to modulation of the melanocortin-4 receptor (MC4R) in needthereof, comprising administering to the subject a pharmaceuticalcomposition comprising: (a) an ionic complex comprising: (i) a cationicpolypeptide represented by the following structural formula:

or a pharmaceutically acceptable salt thereof; and (ii) a1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE) conjugatedpolyethylene glycol, wherein the molar ratio of the cationic polypeptideto the 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE) conjugatedpolyethylene glycol ranges from about 1:1 to about 1:10; and (ii) apharmaceutically acceptable carrier; thereby treating the subject. 87.The method of claim 86, wherein the disease or disorder responsive tomodulation of the MC4R receptor is selected from type 1 diabetes, type 2diabetes, obesity, insulin resistance, metabolic syndrome, male erectiledysfunction, female sexual disorder, non-alcoholic fatty liver disease,non-alcoholic steatohepatitis, disorders of substance abuse, alcoholismfeeding disorders, cachexia, inflammation, Prader-Willy syndrome andanxiety.